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7.21

I Prepare medium

1.soc medium

2.LB medium

3.LBliquid medium

II transform 2011 parts –OUC Order 1 to 3

III pick up a tested single colony

7.22

1．Extract plasmid of the practicing part

2. Measure density using distilled water as comparison

	A260/A280	A260/A230	density（ng/ul）
1	1.892	2.115	0.035
2	1.841	1.227	0.0405
3	2.299	4.054	0.030

3. Digestion for 3 hours

	1	2	3	4(Single enzyme cut)
DNA(ul)	15	15	15	15
Buffer2(ul)	5	5	5	5
Bsa(ul)	0.5	0.5	0.5	-----
Double-distilled water(ul)	27.5	27.5	27.5	29
EcoR I(ul)	1	1	1	1
Pst I(ul)	1	1	1	-------
Time(h)	3	3	3	3

4.check by Electrophoresis

1、2、3：double enzyme，4、5、6：Single enzyme ，7：plasmid

5.transform 7new parts(number:4-10)

6.culture bacteria on LB medium

7.culture 3 new parts in Shaker

7.24

1. Presrvation species of parts No. ,4,5,6,7,8,9,10

2. Extract plasmid of 4,5,6,7,8,9,10

3.check through double enzyme Digestion

7.25

1. Measure density

5	0.020µg/µl
4	0.012µg/µl
6	0.020µg/µl
2	0.015µg/µl

2. Digestion

	5	4	6	2
DNA	25	30	25	25
Buffer2	5	5	5	5
BSA	0.5	0.5	0.5	0.5
H2O	17.5	12.5	17.5	17.5
enzyme1	EcoRⅠ1µl	EcoRⅠ1µl	SpeⅠ1µl	XbaⅠ1µl
Enzyme2	SpeⅠ1µl	XbaⅠ1µl	PstⅠ1µl	PstⅠ1µl

3.check by Electrophoresis

4.gel extraction

5.measure density

OUC-order	5	4	6	2
Density ug/µl	0.016	0.021	0.027	0.028

6. Ligate overnight

	1	2
Parts	2µl 5+3µl 4	3µl 2+3µl 6
Buffer	2µl	2µl
enzyme	1µl	1µl
DdH2O	2µl	1µl

7.28

Repeat the ligation expriments of 7.25

7.29

Extract plasmids of parts No. 11,12,13,14

7.30

Medium has antibiotic resistance problem, re-transform 11\12\13\14

7.31

1.cultrue bacteria of 11,12,13,14 in LB liquid medium

2. Extract plasmid of 15,16,17,18,19,20,21,22

3. Optimization the Steps of Extracting plasmid

4.Chloramphericol preparing

5. NaAC’s DNA concentration

8.1

1. Extract plasmid of 11-14 and Measure density

	ng/µl	A260∕A280
11	12.7	1.917
12	10.3	1.981
13	35.5	1.919
14	15.7	1.975

2. Extract plasmid of No.5,4,6,2，15-20 parts

3.culture the colony of GFP on LB medium

8.2

Measure density of 15-22,2,4,5,6

Parts	µg/µl	A260∕A280
2	0.038	1.875
4	0.019	1.298
5	0.022	1.538
6	0.020	1.702
15	0.012	1.237
16	0.024	1.649
17	0.018	1.731
18	0.015	1.470
21	0.017	1.538

8.3

1. Concentrate plasmid using alcohol and NaAC

2. Measure density after concentrating

	ng/µl	A260∕A280
2	103	1.807
4	33	1.811
5	56	1.836
6	41	1.745

8.4

Digestion

	Double enzymes		Single enzyme
Parts	5	2	4	6
DNA	25	13	27	25
Buffer	5	5	5	5
BSA	0.5	0.5	0.5	0.5
ddH2O	16.5	28.5	16.5	18.5
enzyme	EcoRⅠ 1.5	XbaⅠ1.5	XbaⅠ1	SpeⅠ1
	SpeⅠ1.5	PstⅠ1.5

Parts	4	6
DNA	28.5	30
Buffer	EcoRⅠbuffer 5	Buffer3 5
BSA		0.5
ddH2O	15.5	13.5
enzyme	EcoRⅠ1	PstⅠ1

8.5

1. Begin device 2

2. Design experimental procedures:

3. First construction with parts of device 2

8.6

2+6 successfully ligate

Measure density

Parts	µg/µl	A260∕A280
3	0.085	1.910
16	0.119	1.867
18	0.019	2.033

Measure density after concentrating

Parts	µg/µl	A260∕A280
18	0.0605	1.921
11	0.0335	2.107

Digestion

	Double enzymes		Single enzyme
Parts	18	16	3	11
DNA	20	10	15	29
Buffer	5	5	5	5
BSA	0.5	0.5	0.5	0.5
ddH2O	21.5	31.5	28	14.5
enzyme	EcoRⅠ 1.5	XbaⅠ1.5	XbaⅠ1	SpeⅠ1
	SpeⅠ1.5	PstⅠ1.5

After 8hours

Parts	3	11
DNA	20	20
Buffer	EcoRⅠbuffer 5	Buffer3 5
BSA		0.5
ddH2O	23.5	23
enzyme	EcoRⅠ1	PstⅠ1

8.7

1. Checking 4+5, 2+6 ‘s ligation by Gel electrophoresis

Digestion

μL	2+6	4+5
DNA	2（53ng）	3(28ng)
Buffer2	1	1
BSA	0.1	0.1
ddH2O	6.7	5.9
EcoRⅠ	0.1	0.1
PstⅠ	0.1	0.1

check by small gel

8.8

1.transform the parts of 3-18,11-16,14-19

2. Active bacteria of 4.5.18.11 and culture in LB liquid medium

3. Presrvation species parts of 4-5

8.9

1.check 2+6,4+5 by small gel

2. Measure density

	number	ng/µl	A260∕A280
2+6	1	58	1.902
	2	78.5	1.826
	3	62.5	1.953
	4	60.5	2.017
4+5	1	31	2.081
	2	33	2.089
	3	20.5	2.291

3. Digestion

parts	2+6				5+4
number	1	2	3	4	1	2	3
DNA	2	2	2	2	3	3	5
Buffer3	1	1	1	1	1	1	1
BSA	0.1	0.1	0.1	0.1	0.1	0.1	0.1
ddH2O	6.5	6.5	6.5	6.5	5.5	5.5	5.5
EcoRⅠ	0.2	0.2	0.2	0.2	0.2	0.2	0.2
PstⅠ	0.2	0.2	0.2	0.2	0.2	0.2	0.2

4. Extract plasmid of 4,5,18,11

Measure density

number	ng/µl	A260∕A280
5	73	2.000
4	71.5	2.072
18	32.5	2.305
3	85	1.910
11	95.5	1.950
16	119	1.867

Digestion 50µl

	5	4	18	3	11	16
DNA	13	13	30	11	10	8
Buffer3	5	5	5	5	5	5
BSA	0.5	0.5	0.5	0.5	0.5	0.5
ddH2O	29.5	29.5	19.5	31.5	30.5	34.5
enzyme	EcoRⅠ 1	XbaⅠ1	EcoRⅠ 1	XbaⅠ1	EcoRⅠ 1	XbaⅠ1
	SpeⅠ1	PstⅠ1	SpeⅠ1	PstⅠ1	SpeⅠ1	PstⅠ1

8.11

1. Checking ligation correctness of 14&19 using small gel----fails

2.try to ligate using 3A

8.12

1. Extract plasmid of 5+4 and Measure density

number	ng/µl	A260∕A280
1	55	1.964
2	39	2.324
3	41	2.412
4	81	2.051
5	42	2.154
6	58	1.966

2.check by single enzyme digestion

number	1	2	3	4	5	6
DNA	3	4	4	2	4	3
Buffer EcoR I	1	1	1	1	1	1
ddH2O	5.5	4.5	4.5	6.5	4.5	5.5
EcoRⅠ	0.5	0.5	0.5	0.5	0.5	0.5

Check using small gel

3.cultrue bacteria of part 13

4. ligate 2+6+3,11+16,18+13

8.13

1.Extract plasmid

2.pick colony of 2+6+3,18+3,16+3 and culture in shaker

8.14

Checking whether colony of different size contain different plasmids, or whether could be used to pick colony raising transformation possibility. But it made no difference

8.15

1.measure density

number	µg/µl	A260∕A280
2+6+3-1	0.032	3.439
2+6+3-2	0.064	2.370
11+16	0.056	2.306
11+16	0.102	2.010
18+3	0.037	2.434
18+3	0.033	2.332

2. Digestion

Single enzyme 10µl

number	2+6+3-1	2+6+3-2	3+18-1	3+18-2	11+16	11+16
DNA	4	2	4	6	2.5	1.5
Buffer EcoR I	1	1	1	1	1	1
ddH2O	4.7	6.7	4.7	2.7	6.2	7.2
EcoRⅠ	0.3	0.3	0.3	0.3	0.3	0.3

Double enzyme 10µl

number	18+3-1	18+3-2	18	2+6+3-1	2+6+3-2	2+6	11+16	11+16
DNA	4	6	6	4	2	3	2.5	1.5
Buffer3	1	1	1	1	1	1	1	1
BSA	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
ddH2O	4.3	2.3	2.4	4.3	6.3	5.3	5.8	6.8
EcoRⅠ	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3
PstⅠ	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3

8.16

Check yesterdays’ digestion by gel electrophoresis

Ladder	18-3		18-3		18	6-2-3	6-2-3	6-2
100bp plus	single	double	single	double	double	double	double	double

8.17

1. Presrvation species of pSB1AC3_BBa13017

2. Extract plasmid of 18，pSB1AC3_BBa13017，measure density

number	µg/µl	A260∕A280
pSB1AC3_BBa13017-1	0.199	2.036
pSB1AC3_BBa13017-2	0.168	2.036
pSB1AC3_BBa13017-3	0.113	2.083
pSB1AC3_BBa13017-4	0.103	2.102
pSB1AC3_BBa13017-5	0.174	2.129
pSB1AC3_BBa13017-6	0.147	2.115
pSB1AC3_BBa13017-7	0.064	2.345
pSB1AC3_BBa13017-8	0.106	2.186
pSB1AC3_BBa13017-9	0.163	2.138
pSB1AC3_BBa13017-10	0.170	2.092
18-1	0.110	2.095
18-2	0.124	2.102
18-3	0.140	2.121

3. Prepare medium of TY（culturing rhizobium）,CM（supplemented medium）

4. Digest pSB1AC3_BBa13017 as Carrier

DNA	5
Buffer2	5
BSA	0.5
ddH2O	37.5
EcoRⅠ	1
PstⅠ	1

Extract 3055bp strip

8.18

1.Ligate 5,4

2.transform pSB1C3

8.19 1.culture rhizobium on TY medium

2.prepare CM,M9 medium

3.LeuB PCR

4. Presrvation species of Bean strain

5. Extract plasmid and Digest

number	18	3	2+6	16	11	14+19	13
DNA	12	10	18	9	7	7	6
Buffer2	5	5	5	5	5	5	5
BSA	0.5	0.5	0.5	0.5	0.5	0.5	0.5
ddH2O	30.1	31.5	24.1	33.1	35.1	35.1	36.1
enzyme	EcoRI1.2	XbaⅠ1.2	EcoRI1.2	XbaⅠ1.2	EcoRI1.2	EcoRI1.2	XbaⅠ1.2
	SpeⅠ1.2	PstⅠ1.2	SpeⅠ1.2	PstⅠ1.2	SpeⅠ1.2	SpeⅠ1.2	PstⅠ1.2

8.20

1. Presrvation species of 5+4 and extract plasmid,measure density

number	ng/µl	A260∕A280
1	99.5	2.098
2	140.5	2.162
3	99	2.152
4	123.5	2.093
5	137.5	2.083

2. Digestion

number	1	2	3	4	5
DNA	1.3	1	1.3	1	1
Buffer3	1	1	1	1	1
BSA	0.1	0.1	0.1	0.1	0.1
ddH2O	7.1	7.4	7.1	7.4	7.4
EcoRⅠ	0.25	0.25	0.25	0.25	0.25
PstⅠ	0.25	0.25	0.25	0.25	0.25

8.22

1、Presrvation species of 5+4 and extract plasmid,measure density, digest

2、pick single colonies of 19-14&13；2-6&3 , 11&16 and culture in shaker

3、PCR Leu（50ul），recover and check

10×Taq Buffer(with MgSO4) 5μL

MgCl2 4μL

Primer-F 1.5μL

Primer-R 1.5μL

H2O 33.5μL

dNTP 2μL

DNA 2μL

Taq 0.25μL

Pfu 0.25μL

Recommended By Advicer Tan

Result: success

8.23

1、Presrvation species of 2 6&3，11&16，14 19&13 and extract plasmid, measure density，digest

density

number

2 6&3

11&16

1419&13









density

（ng/ul）

30

49

45

132

62

42

64

Digestion

Single enzyme

	2 6&3	11&16	1419&13				
DNA	3.3	2	2.2	1	1.5	2.3	1.5
Buffer4	1	1	1	1	1	1	1
DdH2O	5.4	6.6	6.4	7.6	7.1	6.3	7.1
XbaI	0.3	0.3	0.3	0.3	0.3	0.3	0.3
BSA	0.1	0.1	0.1	0.1	0.1	0.1	0.1

Double enzymes

	2 6&3	11&16	1419&13				
DNA	3.3	2	2.2	1	1.5	2.3	1.5
Buffer4	1	1	1	1	1	1	1
BSA	0.1	0.1	0.1	0.1	0.1	0.1	0.1
H2O	5.1	6.4	6.2	7.4	6.9	6.1	6.9
EcoRI-HF	0.25	0.25	0.25	0.25	0.25	0.25	0.25
SpeI	0.25	0.25	0.25	0.25	0.25	0.25	0.25

result：success

2、Preservation species of CFN42 WT and BL21

3、check 18&3 be small gel ——succeed

4、Prepare medium：LB liquid medium 300ml，

LB medium 150ml

TY medium 150ml

5、Prepare SMM medium，+ pro+ Vb1

8.24

1、Prepare SMM medium :MM+pro+Vb1 MM+pro+Vb1+leu

2、check by small gel

8.25

1、ligate 11-16&18-13 and 5&4 using 3A

Measure density：

	11-16	18-13	5	4
density（ng/ul）	46.5	136.5	85.5	71

Double enzymes

	5	4	18-3	11-16
DNA	12	10	10	25
Buffer4	5	5	5	5
BSA	0.5	0.5	0.5	0.5
H2O	29.5	32.5	31.5	16.5
enzyme1	EcoRI-HF 1.5	XbaI 1	EcoRI-HF 1.5	XbaI 1.5
enzyme 2	SpeI 1.5	PstI 1	SpeI 1.5	PstI 1.5

Check by gel

Ligation of 5&4

5	4	AC	T4	Buffer	H2O
6	4	4	1	2	3

Ligation of 11-16&18-13：（ul）

K381001	18-3	11-16	Buffer	T4
5	6	6	2	1

2、inoculate HB101 and BL21 into the solid medium (MM+VB1+Pro and MM+VB1+Pro+Leu)，culture the Rhizobium etli CFN42 and Sinorhizobium meliloti 1021 on TY solid medium at 30℃

8.27

1、transform 18-3&11

2、pick 3 colonies of 5&4 and culture in l LB liquid medium

3、culture LeuB and K115001 in LB liquid medium

4、recover 22,21,3 and ligate 21&3,22&3

Ligation of 21&3：(ul)

22	3	T4	Buffer	ddH2O
7	4	1	2	6

Ligation of 22&3：(ul)

21	3	T4	Buffer	ddH2O
8.5	4	1	2	4.5

8.28

1、Prepare medium：LB liquid medium 500ml

2、transform 5&4 and 18-3&11-16 again，and transform leu 21&3 and 22&3

3、 culture deficient cells

4、culture 18-3&11 in LB liquid medium

8.29 1、PCR :5&4 and leu

PCR（ul）

ddH2O	dNTP	Buffer	Primer-F	Primer-R	dna	Taq
39.5	2	5	1	1	1	0.5

2、Presrvation species of 18-3&11 and extract plasmid,measure density, digest

density（1）19.5（2）130.5

digestion

single enzyme

	（1）	（2）
DNA	6	1
Buffer	1	1
BSA	0.1	0.1
ddH2O	2.65	7.65
XbaI	0.35	0.35

Double enzymes:

	（1）	（2）
DNA	6	1
Buffer	1	1
BSA	0.1	0.1
ddH2O	2.4	7.4
EcoRI	0.25	0.25
PstI	0.25	0.25

Check by small gel

3、pick single colony of 5&4，leu,21&3,22&3，18-3&11-16 and culture in LB liquid medium。

4、prepare TY medium 100ml

8.30 1、Presrvation species of 5&4,leuB89,leuB13 and extract plasmid,measure density, digest

density：

leuB13（1）	5&4（1）	5&4（2）	5&4（3）	leuB89（1）	leuB89（2）	leuB89(3)
24.5	188.0	62.0	132.0	6.8	3.1	10.8

Digestion of 5&4

	（1）	（2）	（3）
DNA	1	2	1
Buffer3	1	1	1
ddH2O	7.5	6.5	7.5
BSA	0.1	0.1	0.1
EcoRI	0.2	0.2	0.2
PstI	0.2	0.2	0.2

Check by small gel

Result:5&4 and leuB successfully ligate

8.31

1、did enzyme digestion to 2 part (with psB1AK2 carrier) and K115001 part(with psB1AT3 carrier) and run electrophoresis together with psB1C3. We recycled the carrier part by cutting the gel.

density：

2 part 37.5

K115001 part 120

digestion(ul)

	K115001	2
DNA	9	9
10*H Buffer	2	2
EcoRI	1	1
PstI	1	1
ddH2O	7	7

9.1

1、prepare 0147TY medium

Tryptone 5.0g Yeast extract 3.0g CaCl.6H2O 1.3g Agar 15.0g Distilled water 1.0L

2、ligate 18-3&11-16 and 18-3&11

density：

18-3（1）	18-3（2）	11-16（1）	11-16（2）	K carrier（1）	K carrier（2）	AC（1）	AC(2)
140	98	278.5	203．0	17.5	17.5	7.9	13.1

Digestion:

	18-3	11	11-16
DNA	7	10	4
Buffer	2	2	2
enzyme1	E 1	X 1	X 1
enzyme 2	S 1	P 1	P 1
DdH2O	9	6	12

Ligation:

18-3：3ul 16:1ul 2：6ul T4：1ul Buffer：2ul H2O：7ul

18-3：3ul 11-16：4ul 2：6ul T4：1ul Buffer：2ul ddH2O：4ul

9.2

1、Presrvation species of leuB and extract plasmid,measure density, digest

density：

1	2	3	4
292.5	21.5	223.5	32.0

Digestion:

	leuB	K(pSB1C3)
EcoRI	1	1
PstI	1	1
Buffer	2	2
DNA	14	8.5
ddH2O	2	7.5

Check by small gel:

recover：

2、pick single colony of 18-3&11-16 and 18-3&11 and culture in shaker

3、transform BBa_I751250,as LuxI_AHL producer。Transform BBa_J04450(placI+mRFP)

9.3 1、ligate leuB, K(pSB1C3) carrier and transform

density：

	Before Concentration	After Concentration
LeuB	15	36
pSB1C3	8	25

ligation

LeuB	Psb1c3	T4	Buffer	DdH2O
6	4	1	2	7

2、extract plasmid of 8-3&11-16 and 18-3&11, digest，and check by small gel

Density:

18-3&11-161	18-3&11-162	18-3&11 1	18-3&11 2	5-4（AC）1	5-4（AC） 2	2-6-3
265	242	192	217.5	193	72	63.5

digestion：

	18-3&11-16(1)	18-3&11-16(2)	18-3&11(1)	18-3&11(2)
DNA	3.5	4	5	4.5
Buffer	2	2	2	2
EcoRI	1	1	1	1
PstI	1	1	1	1
ddH2O	12.5	12	11	11.5

Check by gel：

2、ligate 5-4&2-6-3

ligation

K	5-4	2-6-3	T4连接酶	Buffer	DdH2O
6	4	2	1	2	5

9．4

1、transform 5-4&2-6-3，extract plasmid of 2-6-3与2

2、transform Pea Rhizobium

9.5 1、Presrvation species of leuB and extract plasmid,measure density, digest, PCR ,check by gel

PCR

			1	2	3
ddH2O	37.5	37	36.5	36.5	36.5
dNTPmix	4	4	4	4	4
Buffer	5	5	5	5	5
VF2	1.5	1.5	1.5	1.5	1.5
VR	1.5	1.5	1.5	1.5	1.5
tamplate	0	0.5	1	1	1
Taq	0.5	0.5	0.5	0.5	0.5

Check by gel

digestion

	LeuB 1	LeuB 3
DNA	3	3
BufferM	0.5	0.5
ddH2O	1.1	1.1
SpeI	0.2	0.2
XbaI	0.2	0.2

Check by gel

9.6

1、Presrvation species of 54263 and extract plasmid, digest, PCR ,check by gel

digestion

	1	2	3
DNA	1.4	1.4	1.7
Buffer	0.5	0.5	0.5
DdH2O	2.5	2.5	2.2
EcoRI	0.3	0.3	0.3
PstI	0.3	0.3	0.3

9.7

1、measure OD of Deficient cell

	MM（leuB-）	1 leuB+	2 leuB+	3 leuB+	4 leuB+
OD（A340）	1.611-1	1.698-1	1.818-1	1.548-1	1.881-1

2、digest leuB、③、K115001

digestion：

	8	6	LeuB	3	T1	T2	T3
DNA	9	9	10	10	10	13	12
Buffer	2	2	2	2	2	2	2
DdH2O	7	6.6	5.6	5.6	5.6	2.6	3.6
EcoRI	1	1.2	1.2	1.2	1.2	1.2	1.2
SpeI	1	1.2	1.2	1.2	1.2	1.2	1.2

density：

LeuB 98.0

T 1:93.5 2:73.5 3:87

3 1:97 2:73 3:82

9.8 1、extract plasmid of 6，and digest with LeuB

Density of 6：

1	2	3
57	68	50

digestion

	6	6	LeuB
DNA	6	6	10
Buffer	2	2	2
DdH2O	9.6	9.6	5.6
EcoRI	1.2	1.2	1.2
SpeI	1.2	1.2	1.2

2、ligate leuB&3

ligation：

T4	Buffer	leuB	3	T carrier	DdH2O
1	2	3	0.7	10	3.3

9.9

1、check 6, leuB, 8

Gel：

Ligate 8&6、6&leuB

Ligation of 8&6：

T carrier	8	6	T4	Buffer	DdH2O
10	1	1	1	2	5

Ligation of 6&leuB

T carrier	6	LeuB	T4	Buffer	DdH2O
10	1	3	1	2	3

9.10

1、transform 8&6 and leuB

2、culture leuB&3 in LB medium

3、device checking：

Device1 has strong Fluorescent and device 2 hasslight one

Picture:

9.11

1、PCR：sinR、pro-RBS-sinR、raiR、raiR-pro

density

sinI	12.8
SinI pro	9.6
Pro-RBS-sinI	15.1
RaiI	14.2
RaiI pro	9.4
Pro-RBS-raiI	11.4
SinR pro	11.5
Pro-RBS-raiR	15.0

2、pick single colony of 6&leuB

9.12 1、digest 21、22 and ligate

digestion

	21	22
Buffer	2	2
NDA	13.6	15
DdH2O	2	0.6
EcoRI	1.2	1.2
SpeI	1.2	1.2

ligation

21&3

21	3	Buffer	T4	DdH2O	T
3	1	2	1	3	10

22&3

22	3	Buffer	T4	DdH2O	T
5	1	2	1	1	10

2、extract plasmid of leuB-6 and measure density

density：16.5

9．13

1、transform and culture 21&3,22&3,8&6，recover PSB1C3 carrier

2.Pcr

ddH2O	Pfu with MgSO4 buffer	dNTP	PF	PR	Template	Pfu
38µl	5µl	4µl	1µl	1µl	0.5µl	0.5µl

Results：failed

3.digest sinR, sinI,pro- sinI

Digestion:20µl

	sinR	sinI	pro- sinI
DNA	10	11	16
Buffer 10×H	2	2	2
ddH2O	6	5	0
EcoRI	1	1	1
PstI	1	1	1

9.15

Digestion of Leu&3 :20µl

number	1	2	3
DNA(µl)	10	10	9
Buffer 10×H(µl)	2	2	2
ddH2O(µl)	6	6	7
XbaⅠ(µl)	1	1	1
PstⅠ(µl)	1	1	1

9.16

Digest 6 and leub

number	6	Leu
DNA(µl)	15	9
Buffer 10×M(µl)	2	2
ddH2O(µl)	2	7
enzymew	SpeⅠ(µl) 1	XbaⅠ(µl) 1
		PstⅠ(µl) 1

9.18

Ligate Leu and 6 using 3A

	Leu	6	buffer	T4
10µl	2µl	6µl	2µl	1µl

Gel picture：

Digest 6

number	DNA(µl)	Buffer 10× (µl)	PstⅠ(µl)	ddH2O(µl)
6	9	2	1	8

9.20

Measure density

number	ng/µl	A260∕A280
6	179.5	1.851
pSB1C3-1	6.3
pSB1C3-2	8.6	3.510
pSB1C3-3	5.1

Digest Leu

DNA(µl)	Buffer 10×M(µl)	ddH2O(µl)	PstⅠ(µl)	XbaⅠ(µl)
6	2	2	2	8

Ligate Leu& 6

Leu	6	buffer	T4	ddH2O
6	4	2	1	7

9.21

Measure density

number	ng/µl	A260∕A280
raiⅠpro-3	29.5	1.666
raiⅠpro-2	28.5	1.885
raiⅠpro-1	23.0	1.818
C0076	161.0	1.793
E0020e0fp-1	28.5	1.676
E0020e0fp-2	19.5	2.19 Retrieved from "http://2011.igem.org/Team:OUC-China/Result/date" Recent changes What links here Related changes Special pages My preferences Printable version Permanent link Privacy policy Disclaimers

@@ Line 76: / Line 76: @@
                  </ul>
 		<li><a href="/Team:OUC-China/Result/date">Data</a></li>
+              <li><a href="/Team:OUC-China/Result/Puf2011d">Previous Biobricks</a></li>
 	</ul>
 </div>
 <div><p>We've also shared this Data document on Google Docs, <a style="color:green;" href="https://docs.google.com/document/d/106boR2c3uczJ1yOioUxMxTKYvC9hDI8_1T_9dwrY0QA/edit" target="_blank">see here.</a></p></div>
 <div><p class="c2 c22"><span></span></p></div><h1 class="c4"><a name="h.u9me3k1zft2"></a><span>7.21</span></h1><h3 class="c4"><a name="h.5c3btudrepao"></a><span>I &nbsp;Prepare medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>1.soc medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>2.LB medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>3.LBliquid medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>II &nbsp;transform 2011 parts &ndash;OUC Order 1 to 3</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>III &nbsp;pick up a tested single colony</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.tutvix3ykf10"></a><span>7.22</span></h1><h3 class="c4"><a name="h.g2bioc2tx8l7"></a><span>1&#65294;Extract plasmid of the practicing part</span></h3><h3 class="c4"><a name="h.ufnfesh49wep"></a><span>2. Measure density using distilled water as comparison</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c40"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>A260/A280</span></p></td><td class="c13"><p class="c4"><span>A260/A230</span></p></td><td class="c13"><p class="c4"><span>density&#65288;ng/ul&#65289;</span></p></td></tr><tr class="c40"><td class="c5"><p class="c4"><span>1</span></p></td><td class="c5"><p class="c4"><span>1.892</span></p></td><td class="c13"><p class="c4"><span>2.115</span></p></td><td class="c13"><p class="c4"><span>0.035</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>2</span></p></td><td class="c5"><p class="c4"><span>1.841</span></p></td><td class="c13"><p class="c4"><span>1.227</span></p></td><td class="c13"><p class="c4"><span>0.0405</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>3</span></p></td><td class="c5"><p class="c4"><span>2.299</span></p></td><td class="c13"><p class="c4"><span>4.054</span></p></td><td class="c13"><p class="c4"><span>0.030</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.9fgyukkhs06"></a><span>3. Digestion for 3 hours</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c42"><p class="c2"><span></span></p><p class="c2"><span></span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>2</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>4(Single enzyme cut)</span></p><p class="c2"><span></span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>DNA(ul)</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Buffer2(ul)</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Bsa(ul)</span></p></td><td class="c32"><p class="c4"><span>0.5</span></p></td><td class="c32"><p class="c4"><span>0.5</span></p></td><td class="c32"><p class="c4"><span>0.5</span></p></td><td class="c32"><p class="c4"><span>-----</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Double-distilled water(ul)</span></p></td><td class="c32"><p class="c4"><span>27.5</span></p></td><td class="c32"><p class="c4"><span>27.5</span></p></td><td class="c32"><p class="c4"><span>27.5</span></p></td><td class="c32"><p class="c4"><span>29</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>EcoR I(ul)</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Pst I(ul)</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>-------</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Time(h)</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.1ruf9mihj3z2"></a><span>4.check by Electrophoresis</span></h3><p class="c4"><span class="c27">1&#12289;2&#12289;3&#65306;double enzyme&#65292;4&#12289;5&#12289;6&#65306;Single enzyme &#65292;7&#65306;plasmid</span></p><p class="c4"><span class="c27">5.transform 7new parts(number:4-10)</span></p><p class="c4"><span class="c27">6.culture bacteria on LB medium</span></p><p class="c4"><span class="c27">7.culture 3 new parts in Shaker</span></p><h1 class="c4"><a name="h.90bsowdqfwvf"></a><span>7.24</span></h1><h3 class="c4"><a name="h.fllej6viiwbz"></a><span>1. Presrvation species of parts No. ,4,5,6,7,8,9,10</span></h3><h3 class="c4"><a name="h.fllej6viiwbz"></a><span>2. Extract plasmid of 4,5,6,7,8,9,10</span></h3><h3 class="c4"><a name="h.fllej6viiwbz"></a><span>3.check through double enzyme Digestion</span></h3><h3 class="c4 c29"><a name="h.fllej6viiwbz"></a></h3><h1 class="c4"><a name="h.60w0ipkcfych"></a><span>7.25</span></h1><h3 class="c4"><a name="h.r7rj1zpq27u6"></a><span>1. Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c37"><p class="c4"><span>5</span></p></td><td class="c37"><p class="c4"><span>0.020&micro;g/&micro;l</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>4</span></p></td><td class="c37"><p class="c4"><span>0.012&micro;g/&micro;l</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>6</span></p></td><td class="c37"><p class="c4"><span>0.020&micro;g/&micro;l</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>2</span></p></td><td class="c37"><p class="c4"><span>0.015&micro;g/&micro;l</span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><h3 class="c4"><a name="h.kj1r3fgjzq4o"></a><span>2. Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td><td class="c1"><p class="c4"><span>30</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer2</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>H2O</span></p></td><td class="c1"><p class="c4"><span>17.5</span></p></td><td class="c1"><p class="c4"><span>12.5</span></p></td><td class="c1"><p class="c4"><span>17.5</span></p></td><td class="c1"><p class="c4"><span>17.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme1</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1&micro;l</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Enzyme2</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1&micro;l</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.z9jf6h5q8571"></a><span>3.check by Electrophoresis</span></h3><h3 class="c4"><a name="h.z9jf6h5q8571"></a><span>4.gel extraction</span></h3><h3 class="c4"><a name="h.3xuy6enwwjqe"></a><span>5.measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>OUC-order</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Density &nbsp;ug/&micro;l</span></p></td><td class="c1"><p class="c4"><span>0.016</span></p></td><td class="c1"><p class="c4"><span>0.021</span></p></td><td class="c1"><p class="c4"><span>0.027</span></p></td><td class="c1"><p class="c4"><span>0.028</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.7t6dpq2z3uet"></a><span>6. Ligate overnight</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>2&micro;l 5+3&micro;l 4</span></p></td><td class="c9"><p class="c4"><span>3&micro;l 2+3&micro;l 6</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>2&micro;l</span></p></td><td class="c9"><p class="c4"><span>2&micro;l</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzyme</span></p></td><td class="c9"><p class="c4"><span>1&micro;l</span></p></td><td class="c9"><p class="c4"><span>1&micro;l</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DdH2O</span></p></td><td class="c9"><p class="c4"><span>2&micro;l</span></p></td><td class="c9"><p class="c4"><span>1&micro;l</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.v8m6lz1ufk1s"></a><span>7.28</span></h1><h3 class="c4"><a name="h.3yqybicztq70"></a><span>Repeat the ligation expriments of 7.25</span></h3><h1 class="c4"><a name="h.iezpb3pvvzxm"></a><span>7.29</span></h1><h3 class="c4"><a name="h.2tk3jc38wcfz"></a><span>Extract plasmids of parts No. 11,12,13,14</span></h3><h1 class="c4"><a name="h.igfocjjyx4ru"></a><span>7.30</span></h1><h3 class="c4"><a name="h.i6nzwpfyvvjl"></a><span>Medium has antibiotic resistance problem, re-transform 11\12\13\14</span></h3><h1 class="c4"><a name="h.ug8l8jxvj3n1"></a><span>7.31</span></h1><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>1.cultrue bacteria of 11,12,13,14 in LB liquid medium</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>2. Extract plasmid of 15,16,17,18,19,20,21,22</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>3. Optimization the Steps of Extracting plasmid</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>4.Chloramphericol preparing</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>5. NaAC&rsquo;s DNA </span><span><a class="c45" href="http:///t">concentration</a></span></h3><p class="c2"><span><a class="c45" href="http:///t"></a></span></p><h1 class="c4"><a name="h.tfyl7y9pxkmo"></a><span>8.1 </span></h1><h3 class="c4"><a name="h.bx2edw2baey7"></a><span>1. Extract plasmid of 11-14 and Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11</span></p></td><td class="c9"><p class="c4"><span>12.7</span></p></td><td class="c9"><p class="c4"><span>1.917</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>12</span></p></td><td class="c9"><p class="c4"><span>10.3</span></p></td><td class="c9"><p class="c4"><span>1.981</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>13</span></p></td><td class="c9"><p class="c4"><span>35.5</span></p></td><td class="c9"><p class="c4"><span>1.919</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>14</span></p></td><td class="c9"><p class="c4"><span>15.7</span></p></td><td class="c9"><p class="c4"><span>1.975</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.hvilmpnl4guw"></a><span>2. Extract plasmid of No.5,4,6,2&#65292;15-20 parts</span></h3><h3 class="c4"><a name="h.hvilmpnl4guw"></a><span>3.culture the colony of GFP on LB medium</span></h3><h1 class="c4"><a name="h.9dt18ihmbdii"></a><span>8.2</span></h1><h3 class="c4"><a name="h.e15sf2yo8hvd"></a><span>Measure density of 15-22,2,4,5,6</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>0.038</span></p></td><td class="c9"><p class="c4"><span>1.875</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>0.019</span></p></td><td class="c9"><p class="c4"><span>1.298</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>0.022</span></p></td><td class="c9"><p class="c4"><span>1.538</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>0.020</span></p></td><td class="c9"><p class="c4"><span>1.702</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>15</span></p></td><td class="c9"><p class="c4"><span>0.012</span></p></td><td class="c9"><p class="c4"><span>1.237</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>16</span></p></td><td class="c9"><p class="c4"><span>0.024</span></p></td><td class="c9"><p class="c4"><span>1.649</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>17</span></p></td><td class="c9"><p class="c4"><span>0.018</span></p></td><td class="c9"><p class="c4"><span>1.731</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>0.015</span></p></td><td class="c9"><p class="c4"><span>1.470</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>21</span></p></td><td class="c9"><p class="c4"><span>0.017</span></p></td><td class="c9"><p class="c4"><span>1.538</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.i1axlh3f3s5k"></a><span>8.3</span></h1><h3 class="c4"><a name="h.q8al8jclaxs9"></a><span>1. Concentrate plasmid using alcohol and NaAC</span></h3><h3 class="c4"><a name="h.q8al8jclaxs9"></a><span>2. Measure density after concentrating</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>103</span></p></td><td class="c9"><p class="c4"><span>1.807</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>33</span></p></td><td class="c9"><p class="c4"><span>1.811</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>56</span></p></td><td class="c9"><p class="c4"><span>1.836</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>41</span></p></td><td class="c9"><p class="c4"><span>1.745</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.s9gc90q0jnk2"></a><span>8.4</span></h1><h3 class="c4"><a name="h.w2vf5a97h188"></a><span>Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Double enzymes</span></p></td><td class="c1"><p><span></span></p></td><td class="c1"><p class="c4"><span>Single enzyme</span></p></td><td class="c1"><p><span></span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Parts</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td><td class="c1"><p class="c4"><span>13</span></p></td><td class="c1"><p class="c4"><span>27</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>ddH2O</span></p></td><td class="c1"><p class="c4"><span>16.5</span></p></td><td class="c1"><p class="c4"><span>28.5</span></p></td><td class="c1"><p class="c4"><span>16.5</span></p></td><td class="c1"><p class="c4"><span>18.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544; 1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1.5</span></p></td><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c2"><span></span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>28.5</span></p></td><td class="c9"><p class="c4"><span>30</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;buffer 5</span></p></td><td class="c9"><p class="c4"><span>Buffer3 5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>15.5</span></p></td><td class="c9"><p class="c4"><span>13.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzyme</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;1</span></p></td><td class="c9"><p class="c4"><span>Pst&#8544;1</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.hhwrahhkoe43"></a><span>8.5</span></h1><h3 class="c4"><a name="h.t0nrpb7qcvp0"></a><span>1. Begin device 2</span></h3><h3 class="c4"><a name="h.t0nrpb7qcvp0"></a><span>2. Design experimental procedures: </span></h3><h3 class="c4"><a name="h.t0nrpb7qcvp0"></a><span>3. First construction with parts of device 2</span></h3><p class="c2"><span class="c44"></span></p><h1 class="c4"><a name="h.tzzxaggc2un4"></a><span>8.6</span></h1><h3 class="c4"><a name="h.yzze9tnt5vuy"></a><span>2+6 successfully ligate</span></h3><h3 class="c4"><a name="h.8c49qiwztmgq"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>0.085</span></p></td><td class="c9"><p class="c4"><span>1.910</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>16</span></p></td><td class="c9"><p class="c4"><span>0.119</span></p></td><td class="c9"><p class="c4"><span>1.867</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>0.019</span></p></td><td class="c9"><p class="c4"><span>2.033</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.nzti3vckiivz"></a><span>Measure density after concentrating</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>0.0605</span></p></td><td class="c9"><p class="c4"><span>1.921</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11</span></p></td><td class="c9"><p class="c4"><span>0.0335</span></p></td><td class="c9"><p class="c4"><span>2.107</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.ewnnzrlm06qu"></a></h3><h3 class="c4"><a name="h.pf64q93rtmr3"></a><span>Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Double enzymes</span></p></td><td class="c1"><p><span></span></p></td><td class="c1"><p class="c4"><span>Single enzyme</span></p></td><td class="c1"><p><span></span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Parts</span></p></td><td class="c1"><p class="c4"><span>18</span></p></td><td class="c1"><p class="c4"><span>16</span></p></td><td class="c1"><p class="c4"><span>3</span></p></td><td class="c1"><p class="c4"><span>11</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>20</span></p></td><td class="c1"><p class="c4"><span>10</span></p></td><td class="c1"><p class="c4"><span>15</span></p></td><td class="c1"><p class="c4"><span>29</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>ddH2O</span></p></td><td class="c1"><p class="c4"><span>21.5</span></p></td><td class="c1"><p class="c4"><span>31.5</span></p></td><td class="c1"><p class="c4"><span>28</span></p></td><td class="c1"><p class="c4"><span>14.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544; 1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1.5</span></p></td><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c2"><span></span></p></td></tr></tbody></table><h3 class="c4"><a name="h.id7koc7n89jl"></a><span>After 8hours </span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><h3 class="c4"><a name="h.euom5tw4uac7"></a><span>Parts</span></h3></td><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>11</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>20</span></p></td><td class="c9"><p class="c4"><span>20</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;buffer 5</span></p></td><td class="c9"><p class="c4"><span>Buffer3 5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>23.5</span></p></td><td class="c9"><p class="c4"><span>23</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzyme</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;1</span></p></td><td class="c9"><p class="c4"><span>Pst&#8544;1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.ekhxnmiid85t"></a><span>8.7</span></h1><h3 class="c4"><a name="h.zb49mmwonyx9"></a><span>1. Checking 4+5, 2+6 &lsquo;s ligation by Gel electrophoresis</span></h3><h3 class="c4"><a name="h.sts8pafwtw56"></a><span>&nbsp;Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>&mu;L</span></p></td><td class="c9"><p class="c4"><span>2+6</span></p></td><td class="c9"><p class="c4"><span>4+5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>2&#65288;53ng&#65289;</span></p></td><td class="c9"><p class="c4"><span>3(28ng)</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer2</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>6.7</span></p></td><td class="c9"><p class="c4"><span>5.9</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.7hb0wq9fet7i"></a></h3><h3 class="c4"><a name="h.pxbfcxpcw2h0"></a><span>check by small gel</span></h3><h1 class="c49 c4"><a name="h.1n6cx51ccc1m"></a></h1><h1 class="c4"><a name="h.hzg362nyf3j"></a><span>8.8</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.ms09fpm4vjum"></a><span>1.transform the parts of 3-18,11-16,14-19</span></h3><h3 class="c4"><a name="h.ms09fpm4vjum"></a><span>2. Active bacteria of 4.5.18.11 and culture in LB liquid medium</span></h3><h3 class="c4"><a name="h.ms09fpm4vjum"></a><span>3. Presrvation species parts of 4-5</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.fel77li0jvq3"></a><span>8.9</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.golezolfnu2v"></a><span>1.check 2+6,4+5 by small gel </span></h3><h3 class="c4"><a name="h.golezolfnu2v"></a><span>2. Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>number</span></p></td><td class="c13"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c13"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>2+6</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>58</span></p></td><td class="c13"><p class="c4"><span>1.902</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>78.5</span></p></td><td class="c13"><p class="c4"><span>1.826</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>3</span></p></td><td class="c13"><p class="c4"><span>62.5</span></p></td><td class="c13"><p class="c4"><span>1.953</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>4</span></p></td><td class="c13"><p class="c4"><span>60.5</span></p></td><td class="c13"><p class="c4"><span>2.017</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>4+5</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>31</span></p></td><td class="c13"><p class="c4"><span>2.081</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>33</span></p></td><td class="c13"><p class="c4"><span>2.089</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>3</span></p></td><td class="c13"><p class="c4"><span>20.5</span></p></td><td class="c13"><p class="c4"><span>2.291</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.95qz8jl117p"></a><span>3. Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c10"><p class="c4"><span>parts</span></p></td><td class="c10"><p class="c4"><span>2+6</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4"><span>5+4</span></p></td><td class="c16"><p><span></span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>number</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>3</span></p></td><td class="c10"><p class="c4"><span>4</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c16"><p class="c4"><span>2</span></p></td><td class="c16"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>DNA</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>3</span></p></td><td class="c16"><p class="c4"><span>3</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>Buffer3</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c16"><p class="c4"><span>1</span></p></td><td class="c16"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>BSA</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c16"><p class="c4"><span>0.1</span></p></td><td class="c16"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>ddH2O</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>5.5</span></p></td><td class="c16"><p class="c4"><span>5.5</span></p></td><td class="c16"><p class="c4"><span>5.5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.ew5rjvlhlbes"></a></h3><h3 class="c4"><a name="h.ew5rjvlhlbes"></a><span>4. Extract plasmid of &nbsp;4,5,18,11</span></h3><h3 class="c4"><a name="h.fbuct55jz3p2"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><h3 class="c4"><a name="h.hqsj9u31i72y"></a><span>number</span></h3></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>73</span></p></td><td class="c9"><p class="c4"><span>2.000</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>71.5</span></p></td><td class="c9"><p class="c4"><span>2.072</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>32.5</span></p></td><td class="c9"><p class="c4"><span>2.305</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>85</span></p></td><td class="c9"><p class="c4"><span>1.910</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11</span></p></td><td class="c9"><p class="c4"><span>95.5</span></p></td><td class="c9"><p class="c4"><span>1.950</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>16</span></p></td><td class="c9"><p class="c4"><span>119</span></p></td><td class="c9"><p class="c4"><span>1.867</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.3z29ux1deqzo"></a><span>Digestion 50&micro;l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c2"><span></span></p></td><td class="c17"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c17"><p class="c4"><span>18</span></p></td><td class="c21"><p class="c4"><span>3</span></p></td><td class="c21"><p class="c4"><span>11</span></p></td><td class="c21"><p class="c4"><span>16</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA</span></p></td><td class="c17"><p class="c4"><span>13</span></p></td><td class="c21"><p class="c4"><span>13</span></p></td><td class="c17"><p class="c4"><span>30</span></p></td><td class="c21"><p class="c4"><span>11</span></p></td><td class="c21"><p class="c4"><span>10</span></p></td><td class="c21"><p class="c4"><span>8</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer3</span></p></td><td class="c17"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c17"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>BSA</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>29.5</span></p></td><td class="c21"><p class="c4"><span>29.5</span></p></td><td class="c17"><p class="c4"><span>19.5</span></p></td><td class="c21"><p class="c4"><span>31.5</span></p></td><td class="c21"><p class="c4"><span>30.5</span></p></td><td class="c21"><p class="c4"><span>34.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>enzyme</span></p></td><td class="c17"><p class="c4"><span>EcoR&#8544; 1</span></p></td><td class="c21"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c17"><p class="c4"><span>EcoR&#8544; 1</span></p></td><td class="c21"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c21"><p class="c4"><span>EcoR&#8544; 1</span></p></td><td class="c21"><p class="c4"><span>Xba&#8544;1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c2"><span></span></p></td><td class="c17"><p class="c4"><span>Spe&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Pst&#8544;1</span></p></td><td class="c17"><p class="c4"><span>Spe&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Pst&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Spe&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Pst&#8544;1</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.jmqfpq9xkm9z"></a><span>8.11</span></h1><h4 class="c4"><a name="h.m6rgdwzdsd3p"></a><span>1. Checking ligation correctness of 14&amp;19 using small gel----fails</span></h4><h4 class="c4"><a name="h.m6rgdwzdsd3p"></a><span>2.try to ligate using 3A</span></h4><h1 class="c4"><a name="h.z0x6hx6e7ltc"></a><span>8.12</span></h1><h3 class="c4"><a name="h.dm8f9683o5dr"></a><span>1. Extract plasmid of &nbsp;5+4 and Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>55</span></p></td><td class="c9"><p class="c4"><span>1.964</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>39</span></p></td><td class="c9"><p class="c4"><span>2.324</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>41</span></p></td><td class="c9"><p class="c4"><span>2.412</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>81</span></p></td><td class="c9"><p class="c4"><span>2.051</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>42</span></p></td><td class="c9"><p class="c4"><span>2.154</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>58</span></p></td><td class="c9"><p class="c4"><span>1.966</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.77mylrq8vnt2"></a><span>2.check by single enzyme digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>number</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>2</span></p></td><td class="c17"><p class="c4"><span>3</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA</span></p></td><td class="c17"><p class="c4"><span>3</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c17"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>2</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer EcoR I</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>5.5</span></p></td><td class="c21"><p class="c4"><span>4.5</span></p></td><td class="c17"><p class="c4"><span>4.5</span></p></td><td class="c21"><p class="c4"><span>6.5</span></p></td><td class="c21"><p class="c4"><span>4.5</span></p></td><td class="c21"><p class="c4"><span>5.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.sp1o9aob8jry"></a></h3><h3 class="c4"><a name="h.f568ba30zhy7"></a><span>Check using small gel</span></h3><h3 class="c4"><a name="h.f568ba30zhy7"></a><span>3.cultrue bacteria of part 13</span></h3><h3 class="c4"><a name="h.f568ba30zhy7"></a><span>4. ligate &nbsp;2+6+3,11+16,18+13</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.alf83syip56z"></a><span>8.13</span></h1><h3 class="c4"><a name="h.ke3ebgbum5n"></a><span>1.Extract plasmid </span></h3><h3 class="c4"><a name="h.ke3ebgbum5n"></a><span>2.pick colony of 2+6+3,18+3,16+3 and culture in shaker</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.e0ipvrwodbd7"></a><span>8.14 </span></h1><h4 class="c4"><a name="h.qq0p6vx7yo2i"></a><span>Checking whether colony of different size contain different plasmids, or whether could be used to pick colony raising transformation possibility. But it made no difference</span></h4><h1 class="c4"><a name="h.5k95r8qmtjok"></a><span>8.15</span></h1><h3 class="c4"><a name="h.wy8969l7pop"></a><span>1.measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2+6+3-1</span></p></td><td class="c9"><p class="c4"><span>0.032</span></p></td><td class="c9"><p class="c4"><span>3.439</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2+6+3-2</span></p></td><td class="c9"><p class="c4"><span>0.064</span></p></td><td class="c9"><p class="c4"><span>2.370</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11+16</span></p></td><td class="c9"><p class="c4"><span>0.056</span></p></td><td class="c9"><p class="c4"><span>2.306</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11+16</span></p></td><td class="c9"><p class="c4"><span>0.102</span></p></td><td class="c9"><p class="c4"><span>2.010</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18+3</span></p></td><td class="c9"><p class="c4"><span>0.037</span></p></td><td class="c9"><p class="c4"><span>2.434</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18+3</span></p></td><td class="c9"><p class="c4"><span>0.033</span></p></td><td class="c9"><p class="c4"><span>2.332</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.3m4xfb4u4qv8"></a><span>2. Digestion</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.c1ekew4tc9t5"></a><span>Single enzyme 10&micro;l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>number</span></p></td><td class="c17"><p class="c4"><span>2+6+3-1</span></p></td><td class="c21"><p class="c4"><span>2+6+3-2</span></p></td><td class="c17"><p class="c4"><span>3+18-1</span></p></td><td class="c21"><p class="c4"><span>3+18-2</span></p></td><td class="c21"><p class="c4"><span>11+16</span></p></td><td class="c21"><p class="c4"><span>11+16</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA</span></p></td><td class="c17"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>2</span></p></td><td class="c17"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>6</span></p></td><td class="c21"><p class="c4"><span>2.5</span></p></td><td class="c21"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer EcoR I</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>4.7</span></p></td><td class="c21"><p class="c4"><span>6.7</span></p></td><td class="c17"><p class="c4"><span>4.7</span></p></td><td class="c21"><p class="c4"><span>2.7</span></p></td><td class="c21"><p class="c4"><span>6.2</span></p></td><td class="c21"><p class="c4"><span>7.2</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c17"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td><td class="c17"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.apo7y8k82owr"></a><span>Double enzyme 10&micro;l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c11"><p class="c4"><span>number</span></p></td><td class="c20"><p class="c4"><span>18+3-1</span></p></td><td class="c11"><p class="c4"><span>18+3-2</span></p></td><td class="c11"><p class="c4"><span>18</span></p></td><td class="c11"><p class="c4"><span>2+6+3-1</span></p></td><td class="c20"><p class="c4"><span>2+6+3-2</span></p></td><td class="c20"><p class="c4"><span>2+6</span></p></td><td class="c20"><p class="c4"><span>11+16</span></p></td><td class="c20"><p class="c4"><span>11+16</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>DNA</span></p></td><td class="c20"><p class="c4"><span>4</span></p></td><td class="c11"><p class="c4"><span>6</span></p></td><td class="c11"><p class="c4"><span>6</span></p></td><td class="c11"><p class="c4"><span>4</span></p></td><td class="c20"><p class="c4"><span>2</span></p></td><td class="c20"><p class="c4"><span>3</span></p></td><td class="c20"><p class="c4"><span>2.5</span></p></td><td class="c20"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>Buffer3</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c11"><p class="c4"><span>1</span></p></td><td class="c11"><p class="c4"><span>1</span></p></td><td class="c11"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>BSA</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c11"><p class="c4"><span>0.1</span></p></td><td class="c11"><p class="c4"><span>0.1</span></p></td><td class="c11"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>ddH2O</span></p></td><td class="c20"><p class="c4"><span>4.3</span></p></td><td class="c11"><p class="c4"><span>2.3</span></p></td><td class="c11"><p class="c4"><span>2.4</span></p></td><td class="c11"><p class="c4"><span>4.3</span></p></td><td class="c20"><p class="c4"><span>6.3</span></p></td><td class="c20"><p class="c4"><span>5.3</span></p></td><td class="c20"><p class="c4"><span>5.8</span></p></td><td class="c20"><p class="c4"><span>6.8</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.l40msbsdoxxo"></a><span>8.16</span></h1><h3 class="c4"><a name="h.9a0lm2k3wig9"></a><span>Check yesterdays&rsquo; digestion by gel electrophoresis</span></h3><p class="c4"><img height="573" src="images/image00.jpg" width="573"></p><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c47"><p class="c4 c22"><span>Ladder</span></p></td><td class="c48"><p class="c4 c22"><span>18-3</span></p></td><td class="c47"><p><span></span></p></td><td class="c47"><p class="c4 c22"><span>18-3</span></p></td><td class="c47"><p><span></span></p></td><td class="c48"><p class="c4 c22"><span>18</span></p></td><td class="c48"><p class="c4 c22"><span>6-2-3</span></p></td><td class="c48"><p class="c4 c22"><span>6-2-3</span></p></td><td class="c48"><p class="c4 c22"><span>6-2</span></p></td></tr><tr class="c12"><td class="c47"><p class="c4 c22"><span>100bp plus</span></p></td><td class="c48"><p class="c4 c22"><span>single</span></p></td><td class="c47"><p class="c4 c22"><span>double</span></p></td><td class="c47"><p class="c4 c22"><span>single</span></p></td><td class="c47"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.6vm4ek23k6ec"></a><span>8.17</span></h1><h3 class="c4"><a name="h.t0sbfske940j"></a><span>1. Presrvation species of pSB1AC3_BBa13017</span></h3><h3 class="c4"><a name="h.n83ej944zlf8"></a><span>2. Extract plasmid of 18&#65292;pSB1AC3_BBa13017&#65292;measure density </span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-1</span></p></td><td class="c9"><p class="c4"><span>0.199</span></p></td><td class="c9"><p class="c4"><span>2.036</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-2</span></p></td><td class="c9"><p class="c4"><span>0.168</span></p></td><td class="c9"><p class="c4"><span>2.036</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-3</span></p></td><td class="c9"><p class="c4"><span>0.113</span></p></td><td class="c9"><p class="c4"><span>2.083</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-4</span></p></td><td class="c9"><p class="c4"><span>0.103</span></p></td><td class="c9"><p class="c4"><span>2.102</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-5</span></p></td><td class="c9"><p class="c4"><span>0.174</span></p></td><td class="c9"><p class="c4"><span>2.129</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-6</span></p></td><td class="c9"><p class="c4"><span>0.147</span></p></td><td class="c9"><p class="c4"><span>2.115</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-7</span></p></td><td class="c9"><p class="c4"><span>0.064</span></p></td><td class="c9"><p class="c4"><span>2.345</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-8</span></p></td><td class="c9"><p class="c4"><span>0.106</span></p></td><td class="c9"><p class="c4"><span>2.186</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-9</span></p></td><td class="c9"><p class="c4"><span>0.163</span></p></td><td class="c9"><p class="c4"><span>2.138</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-10</span></p></td><td class="c9"><p class="c4"><span>0.170</span></p></td><td class="c9"><p class="c4"><span>2.092</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18-1</span></p></td><td class="c9"><p class="c4"><span>0.110</span></p></td><td class="c9"><p class="c4"><span>2.095</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18-2</span></p></td><td class="c9"><p class="c4"><span>0.124</span></p></td><td class="c9"><p class="c4"><span>2.102</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18-3</span></p></td><td class="c9"><p class="c4"><span>0.140</span></p></td><td class="c9"><p class="c4"><span>2.121</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.h74qm3ioftn6"></a><span>3. Prepare medium &nbsp;of &nbsp;TY&#65288;culturing rhizobium&#65289;,CM&#65288;supplemented medium&#65289;</span></h3><h3 class="c4"><a name="h.h74qm3ioftn6"></a><span>4. Digest pSB1AC3_BBa13017 as Carrier</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c37"><p class="c4"><span>DNA</span></p></td><td class="c37"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Buffer2</span></p></td><td class="c37"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>BSA</span></p></td><td class="c37"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>ddH2O</span></p></td><td class="c37"><p class="c4"><span>37.5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c37"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c37"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c4"><img height="610" src="images/image01.jpg" width="610"></p><h6 class="c4"><a name="h.jnl1gq2qm9xp"></a><span>Extract 3055bp strip</span></h6><h1 class="c4"><a name="h.bgliaogofoy0"></a><span>8.18</span></h1><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>1.Ligate 5,4</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>2.transform &nbsp;pSB1C3</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>8.19</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>1.culture rhizobium on TY medium</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>2.prepare CM,M9 medium</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>3.LeuB PCR</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>4. Presrvation species of Bean strain</span></h3><h3 class="c4"><a name="h.wuw8mhckhe1z"></a><span>5. Extract plasmid and Digest</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c10"><p class="c4"><span>number</span></p></td><td class="c10"><p class="c4"><span>18</span></p></td><td class="c10"><p class="c4"><span>3</span></p></td><td class="c10"><p class="c4"><span>2+6</span></p></td><td class="c10"><p class="c4"><span>16</span></p></td><td class="c10"><p class="c4"><span>11</span></p></td><td class="c16"><p class="c4"><span>14+19</span></p></td><td class="c16"><p class="c4"><span>13</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>DNA</span></p></td><td class="c10"><p class="c4"><span>12</span></p></td><td class="c10"><p class="c4"><span>10</span></p></td><td class="c10"><p class="c4"><span>18</span></p></td><td class="c10"><p class="c4"><span>9</span></p></td><td class="c10"><p class="c4"><span>7</span></p></td><td class="c16"><p class="c4"><span>7</span></p></td><td class="c16"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>Buffer2</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>BSA</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c16"><p class="c4"><span>0.5</span></p></td><td class="c16"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>ddH2O</span></p></td><td class="c10"><p class="c4"><span>30.1</span></p></td><td class="c10"><p class="c4"><span>31.5</span></p></td><td class="c10"><p class="c4"><span>24.1</span></p></td><td class="c10"><p class="c4"><span>33.1</span></p></td><td class="c10"><p class="c4"><span>35.1</span></p></td><td class="c16"><p class="c4"><span>35.1</span></p></td><td class="c16"><p class="c4"><span>36.1</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>enzyme</span></p></td><td class="c10"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c10"><p class="c4"><span>Xba&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c10"><p class="c4"><span>Xba&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c16"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c16"><p class="c4"><span>Xba&#8544;1.2</span></p></td></tr><tr class="c12"><td class="c10"><p class="c2"><span></span></p></td><td class="c10"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Pst&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Pst&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c16"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c16"><p class="c4"><span>Pst&#8544;1.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.79kmvmm53rae"></a><span>8.20</span></h1><h3 class="c4"><a name="h.rb7ae2jhzsxo"></a><span>1. Presrvation species of 5+4 and extract plasmid,measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>99.5</span></p></td><td class="c9"><p class="c4"><span>2.098</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>140.5</span></p></td><td class="c9"><p class="c4"><span>2.162</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>99</span></p></td><td class="c9"><p class="c4"><span>2.152</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>123.5</span></p></td><td class="c9"><p class="c4"><span>2.093</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>137.5</span></p></td><td class="c9"><p class="c4"><span>2.083</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.cd31d7gjoaj9"></a><span>2. Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>number</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>DNA</span></p></td><td class="c0"><p class="c4"><span>1.3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1.3</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Buffer3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>BSA</span></p></td><td class="c0"><p class="c4"><span>0.1</span></p></td><td class="c0"><p class="c4"><span>0.1</span></p></td><td class="c0"><p class="c4"><span>0.1</span></p></td><td class="c6"><p class="c4"><span>0.1</span></p></td><td class="c6"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>ddH2O</span></p></td><td class="c0"><p class="c4"><span>7.1</span></p></td><td class="c0"><p class="c4"><span>7.4</span></p></td><td class="c0"><p class="c4"><span>7.1</span></p></td><td class="c6"><p class="c4"><span>7.4</span></p></td><td class="c6"><p class="c4"><span>7.4</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c49 c4"><a name="h.wlf6j8rjal3t"></a></h1><h1 class="c4"><a name="h.urk4as897tek"></a><span>8.22</span></h1><h3 class="c4"><a name="h.dwwnj6olsp9f"></a><span>1&#12289;Presrvation species of 5+4 and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.4k6v2rq1fzij"></a><span>2&#12289;pick single colonies of 19-14&amp;13&#65307;2-6&amp;3 , 11&amp;16 and culture in shaker</span></h3><h3 class="c4"><a name="h.k3thznec7nhf"></a><span>3&#12289;PCR Leu&#65288;50ul&#65289;&#65292;recover and check</span></h3><p class="c2"><span></span></p><p class="c4"><img height="197" src="images/image04.jpg" width="187"></p><p class="c4"><span>1</span><span class="c3">0&times;</span><span>Taq Buffer(with MgSO4) 5&mu;L</span></p><p class="c4"><span>MgCl2 &nbsp;4&mu;L</span></p><p class="c4"><span>Primer-F 1.5&mu;L</span></p><p class="c4"><span>Primer-R 1.5&mu;L</span></p><p class="c4"><span>H2O 33.5&mu;L</span></p><p class="c4"><span>dNTP 2&mu;L</span></p><p class="c4"><span>DNA 2&mu;L</span></p><p class="c4"><span>Taq 0.25&mu;L</span></p><p class="c4"><span>Pfu 0.25&mu;L &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span></p><p class="c4 c22"><span>Recommended By Advicer Tan</span></p><p class="c4 c22"><span>Result: success</span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><h1 class="c49 c4"><a name="h.2yfk6buo0k2e"></a></h1><h1 class="c4"><a name="h.2yfk6buo0k2e"></a><span>8.23</span></h1><h3 class="c4"><a name="h.g493ims3jbp8"></a><span>1&#12289;Presrvation species of 2 6&amp;3&#65292;11&amp;16&#65292;14 19&amp;13 and extract plasmid, measure density&#65292;digest</span></h3><h3 class="c4"><a name="h.tknykw38j7c4"></a><span>density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c15"><p class="c4"><span>number</span></p></td><td class="c30"><p class="c4"><span>2 6&amp;3</span></p></td><td class="c54"><p class="c4"><span>11&amp;16</span></p></td><td class="c51"><p class="c4"><span>1419&amp;13</span><span class="c3">&#61569;</span></p></td><td class="c41"><p class="c4"><span class="c3">&#61570;</span></p></td><td class="c30"><p class="c4"><span class="c3">&#61571;</span></p></td><td class="c39"><p class="c4"><span class="c3">&#61572;</span></p></td><td class="c39"><p class="c4"><span class="c3">&#61573;</span></p></td></tr><tr class="c12"><td class="c15"><p class="c4"><span>density</span></p><p class="c4"><span>&#65288;ng/ul&#65289;</span></p></td><td class="c30"><p class="c4"><span>30</span></p></td><td class="c54"><p class="c4"><span>49</span></p></td><td class="c51"><p class="c4"><span>45</span></p></td><td class="c41"><p class="c4"><span>132</span></p></td><td class="c30"><p class="c4"><span>62</span></p></td><td class="c39"><p class="c4"><span>42</span></p></td><td class="c39"><p class="c4"><span>64</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.r1xl2xgbvvgi"></a><span>Digestion</span></h3><h3 class="c4"><a name="h.qvlzmnr7s30"></a><span>Single enzyme</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c30"><p class="c2"><span></span></p></td><td class="c31"><p class="c4"><span>2 6&amp;3</span></p></td><td class="c30"><p class="c4"><span>11&amp;16</span></p></td><td class="c35"><p class="c4"><span>1419&amp;13</span><span class="c3">&#61569;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61570;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61571;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61572;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61573;</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>DNA</span></p></td><td class="c31"><p class="c4"><span>3.3</span></p></td><td class="c30"><p class="c4"><span>2</span></p></td><td class="c35"><p class="c4"><span>2.2</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td><td class="c8"><p class="c4"><span>2.3</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>Buffer4</span></p></td><td class="c31"><p class="c4"><span>1</span></p></td><td class="c30"><p class="c4"><span>1</span></p></td><td class="c35"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>DdH2O</span></p></td><td class="c31"><p class="c4"><span>5.4</span></p></td><td class="c30"><p class="c4"><span>6.6</span></p></td><td class="c35"><p class="c4"><span>6.4</span></p></td><td class="c8"><p class="c4"><span>7.6</span></p></td><td class="c8"><p class="c4"><span>7.1</span></p></td><td class="c8"><p class="c4"><span>6.3</span></p></td><td class="c8"><p class="c4"><span>7.1</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>XbaI</span></p></td><td class="c31"><p class="c4"><span>0.3</span></p></td><td class="c30"><p class="c4"><span>0.3</span></p></td><td class="c35"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>BSA</span></p></td><td class="c31"><p class="c4"><span>0.1</span></p></td><td class="c30"><p class="c4"><span>0.1</span></p></td><td class="c35"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.78jdujd9b23x"></a><span>Double enzymes</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c33"><p class="c2"><span></span></p></td><td class="c8"><p class="c4"><span>2 6&amp;3</span></p></td><td class="c25"><p class="c4"><span>11&amp;16</span></p></td><td class="c35"><p class="c4"><span>1419&amp;13</span><span class="c3">&#61569;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61570;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61571;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61572;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61573;</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>DNA</span></p></td><td class="c8"><p class="c4"><span>3.3</span></p></td><td class="c25"><p class="c4"><span>2</span></p></td><td class="c35"><p class="c4"><span>2.2</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td><td class="c8"><p class="c4"><span>2.3</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>Buffer4</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c25"><p class="c4"><span>1</span></p></td><td class="c35"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>BSA</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c25"><p class="c4"><span>0.1</span></p></td><td class="c35"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>H2O</span></p></td><td class="c8"><p class="c4"><span>5.1</span></p></td><td class="c25"><p class="c4"><span>6.4</span></p></td><td class="c35"><p class="c4"><span>6.2</span></p></td><td class="c8"><p class="c4"><span>7.4</span></p></td><td class="c8"><p class="c4"><span>6.9</span></p></td><td class="c8"><p class="c4"><span>6.1</span></p></td><td class="c8"><p class="c4"><span>6.9</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>EcoRI-HF</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c25"><p class="c4"><span>0.25</span></p></td><td class="c35"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>SpeI</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c25"><p class="c4"><span>0.25</span></p></td><td class="c35"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.uosio89ahtai"></a><span>result&#65306;success</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>2&#12289;</span><span class="c36">Preservation</span><span>&nbsp;species of CFN42 WT and BL21</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>3&#12289;check 18&amp;3 be small gel &mdash;&mdash;succeed</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>4&#12289;Prepare medium&#65306;LB liquid medium &nbsp;300ml&#65292;</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>LB medium 150ml </span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;TY medium 150ml</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>5&#12289;Prepare SMM medium&#65292;+ pro+ Vb1</span></h3><h1 class="c4"><a name="h.r6ryyomviv4b"></a><span>8.24</span></h1><h3 class="c4"><a name="h.ciewiyq5t66a"></a><span>1&#12289;Prepare SMM medium &nbsp;:MM+pro+Vb1 &nbsp;MM+pro+Vb1+leu</span></h3><h3 class="c4"><a name="h.ciewiyq5t66a"></a><span>2&#12289;check by small gel</span></h3><h1 class="c4"><a name="h.he4e0lgvyqrw"></a><span>8.25</span></h1><h3 class="c4"><a name="h.8kqf064rpjw5"></a><a href="#" name="id.b6b2514dfea5"></a><span>1&#12289;ligate 11-16&amp;18-13 and 5&amp;4 using 3A</span></h3><h3 class="c4"><a name="h.tkxpq18ksh8e"></a><span>Measure density&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>11-16</span></p></td><td class="c1"><p class="c4"><span>18-13</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>density&#65288;ng/ul&#65289;</span></p></td><td class="c1"><p class="c4"><span>46.5</span></p></td><td class="c1"><p class="c4"><span>136.5</span></p></td><td class="c1"><p class="c4"><span>85.5</span></p></td><td class="c1"><p class="c4"><span>71</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.f8muhyhuv6l2"></a><span>Double enzymes</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>18-3</span></p></td><td class="c1"><p class="c4"><span>11-16</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>12</span></p></td><td class="c1"><p class="c4"><span>10</span></p></td><td class="c1"><p class="c4"><span>10</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer4</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>H2O</span></p></td><td class="c1"><p class="c4"><span>29.5</span></p></td><td class="c1"><p class="c4"><span>32.5</span></p></td><td class="c1"><p class="c4"><span>31.5</span></p></td><td class="c1"><p class="c4"><span>16.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme1</span></p></td><td class="c1"><p class="c4"><span>EcoRI-HF 1.5</span></p></td><td class="c1"><p class="c4"><span>XbaI &nbsp;1</span></p></td><td class="c1"><p class="c4"><span>EcoRI-HF &nbsp;1.5</span></p></td><td class="c1"><p class="c4"><span>XbaI &nbsp;1.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme 2</span></p></td><td class="c1"><p class="c4"><span>SpeI 1.5</span></p></td><td class="c1"><a href="#" name="id.5ffd2b59663f"></a><p class="c4"><span>PstI &nbsp;1</span></p></td><td class="c1"><p class="c4"><span>SpeI &nbsp;1.5</span></p></td><td class="c1"><p class="c4"><span>PstI &nbsp;1.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ynposbrw4sji"></a><span>Check by gel</span></h3><h3 class="c4"><a name="h.sundqa7jgp4t"></a><span>Ligation of 5&amp;4</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>AC</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>H2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ezohoz16dnao"></a><span>Ligation of &nbsp;11-16&amp;18-13&#65306;&#65288;ul&#65289;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>K381001</span></p></td><td class="c0"><p class="c4"><span>18-3</span></p></td><td class="c0"><p class="c4"><span>11-16</span></p></td><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>T4</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.8ylqa0m2w31v"></a><span>2&#12289;inoculate HB101 and BL21 into the solid medium (MM+VB1+Pro and MM+VB1+Pro+Leu)&#65292;culture the Rhizobium etli CFN42 and Sinorhizobium meliloti 1021 on TY solid medium at 30&#8451;</span></h3><h3 class="c29 c4"><a name="h.94y273j4x26h"></a></h3><h1 class="c4"><a name="h.b25s7bsf4qft"></a><span>8.27</span></h1><h3 class="c4"><a name="h.94y273j4x26h"></a><span>1&#12289;transform 18-3&amp;11</span></h3><h3 class="c4"><a name="h.94y273j4x26h"></a><span>2&#12289;pick 3 colonies of 5&amp;4 and culture in l LB liquid medium</span></h3><h3 class="c4"><a name="h.94y273j4x26h"></a><span>3&#12289;culture LeuB and K115001 in LB liquid medium</span></h3><h3 class="c4"><a name="h.94y273j4x26h"></a><span>4&#12289;recover 22,21,3 and ligate 21&amp;3,22&amp;3</span></h3><h3 class="c4"><a name="h.k3ru3lgh65ye"></a><a href="#" name="id.887d11515b76"></a><span>Ligation of 21&amp;3&#65306;(ul)</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>22</span></p></td><td class="c1"><p class="c4"><span>3</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>ddH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>7</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.6pt8yxfqhwg6"></a></h3><h3 class="c4"><a name="h.6pt8yxfqhwg6"></a><span>Ligation of &nbsp;22&amp;3&#65306;(ul)</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>21</span></p></td><td class="c1"><p class="c4"><span>3</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>ddH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>8.5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>4.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><h1 class="c4"><a name="h.5bliw8b6jufq"></a><span>8.28</span></h1><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>1&#12289;Prepare medium&#65306;LB liquid medium &nbsp;500ml</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>2&#12289;transform 5&amp;4 and 18-3&amp;11-16 again&#65292;and transform leu 21&amp;3 and 22&amp;3</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>3&#12289; culture deficient cells</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>4&#12289;culture 18-3&amp;11 in LB liquid medium </span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>8.29</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>1&#12289;PCR :5&amp;4 and leu</span></h3><h3 class="c4"><a name="h.awlrj81666pu"></a><span>PCR&#65288;ul&#65289;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>dNTP</span></p></td><td class="c21"><p class="c4"><span>Buffer</span></p></td><td class="c17"><a href="#" name="id.bd219e189f6f"></a><p class="c4"><span>Primer-F</span></p></td><td class="c21"><p class="c4"><span>Primer-R</span></p></td><td class="c21"><p class="c4"><span>dna</span></p></td><td class="c21"><p class="c4"><span>Taq</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>39.5</span></p></td><td class="c17"><p class="c4"><span>2</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.cbhr3fgm4nrj"></a><span>2&#12289;Presrvation species of 18-3&amp;11 and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.cbhr3fgm4nrj"></a><span>density&#65288;1&#65289;19.5&#65288;2&#65289;130.5</span></h3><h3 class="c4"><a name="h.cbhr3fgm4nrj"></a><span>digestion</span></h3><h3 class="c4"><a name="h.p91mu85t6yz4"></a><span>single enzyme</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>&#65288;1&#65289;</span></p></td><td class="c9"><p class="c4"><span>&#65288;2&#65289;</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>2.65</span></p></td><td class="c9"><p class="c4"><span>7.65</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>XbaI</span></p></td><td class="c9"><p class="c4"><span>0.35</span></p></td><td class="c9"><p class="c4"><span>0.35</span></p></td></tr></tbody></table><p class="c4"><span>Double enzymes:</span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>&#65288;1&#65289;</span></p></td><td class="c9"><p class="c4"><span>&#65288;2&#65289;</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>2.4</span></p></td><td class="c9"><p class="c4"><span>7.4</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoRI</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>PstI</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.71fxa3curofb"></a><span>Check by small gel</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>3&#12289;pick single colony of 5&amp;4&#65292;leu,21&amp;3,22&amp;3&#65292;18-3&amp;11-16 and culture in LB liquid medium&#12290;</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>4&#12289;prepare TY medium 100ml</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>8.30</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><a href="#" name="id.e846d4e866ce"></a><span>1&#12289;Presrvation species of 5&amp;4,leuB89,leuB13 and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>leuB13&#65288;1&#65289;</span></p></td><td class="c17"><a href="#" name="id.f54c4c23a77f"></a><p class="c4"><span>5&amp;4&#65288;1&#65289;</span></p></td><td class="c21"><p class="c4"><span>5&amp;4&#65288;2&#65289;</span></p></td><td class="c17"><p class="c4"><span>5&amp;4&#65288;3&#65289;</span></p></td><td class="c21"><p class="c4"><span>leuB89&#65288;1&#65289;</span></p></td><td class="c21"><p class="c4"><span>leuB89&#65288;2&#65289;</span></p></td><td class="c21"><p class="c4"><span>leuB89(3)</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>24.5</span></p></td><td class="c17"><p class="c4"><span>188.0</span></p></td><td class="c21"><p class="c4"><span>62.0</span></p></td><td class="c17"><p class="c4"><span>132.0</span></p></td><td class="c21"><p class="c4"><span>6.8</span></p></td><td class="c21"><p class="c4"><span>3.1</span></p></td><td class="c21"><p class="c4"><span>10.8</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.6jtrl26b1tlv"></a><span>Digestion of &nbsp;5&amp;4</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>&#65288;1&#65289;</span></p></td><td class="c13"><p class="c4"><span>&#65288;2&#65289;</span></p></td><td class="c13"><p class="c4"><span>&#65288;3&#65289;</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer3</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>ddH2O</span></p></td><td class="c5"><p class="c4"><span>7.5</span></p></td><td class="c13"><p class="c4"><span>6.5</span></p></td><td class="c13"><p class="c4"><span>7.5</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>BSA</span></p></td><td class="c5"><p class="c4"><span>0.1</span></p></td><td class="c13"><p class="c4"><span>0.1</span></p></td><td class="c13"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI</span></p></td><td class="c5"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.9sbx477gdv2a"></a><span>Check by small gel</span></h3><h3 class="c4"><a name="h.9sbx477gdv2a"></a><span>Result:5&amp;4 and leuB successfully ligate</span></h3><h1 class="c4"><a name="h.em1ctnutxs3l"></a><span>8.31</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>1&#12289;did enzyme digestion to 2 part (with psB1AK2 carrier) and K115001 part(with psB1AT3 carrier) and run electrophoresis together with psB1C3. We recycled the carrier part by cutting the gel.</span></h3><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>density&#65306;</span></h3><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>2 part &nbsp; 37.5</span></h3><a href="#" name="id.a5fc2f8264e3"></a><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>K115001 part &nbsp; 120</span></h3><h3 class="c4"><a name="h.p7f0i2i3brxd"></a><span>digestion(ul)</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>K115001</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>9</span></p></td><td class="c9"><p class="c4"><span>9</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>10*H Buffer</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoRI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>PstI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>7</span></p></td><td class="c9"><p class="c4"><span>7</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.ox56qgmq045h"></a><span>9.1</span></h1><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>1&#12289;prepare 0147TY medium</span></h3><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>Tryptone &nbsp;5.0g &nbsp;Yeast extract 3.0g &nbsp;CaCl.6H2O &nbsp;1.3g &nbsp;Agar &nbsp;15.0g Distilled water 1.0L</span></h3><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>2&#12289;ligate 18-3&amp;11-16 and 18-3&amp;11</span></h3><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c10"><p class="c4"><span>18-3&#65288;1&#65289;</span></p></td><td class="c10"><p class="c4"><span>18-3&#65288;2&#65289;</span></p></td><td class="c10"><p class="c4"><span>11-16&#65288;1&#65289;</span></p></td><td class="c10"><p class="c4"><span>11-16&#65288;2&#65289;</span></p></td><td class="c10"><p class="c4"><span>K carrier&#65288;1&#65289;</span></p></td><td class="c10"><p class="c4"><span>K carrier&#65288;2&#65289;</span></p></td><td class="c16"><p class="c4"><span>AC&#65288;1&#65289;</span></p></td><td class="c16"><p class="c4"><span>AC(2)</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>140</span></p></td><td class="c10"><p class="c4"><span>98</span></p></td><td class="c10"><p class="c4"><span>278.5</span></p></td><td class="c10"><p class="c4"><span>203&#65294;0</span></p></td><td class="c10"><p class="c4"><span>17.5</span></p></td><td class="c10"><p class="c4"><span>17.5</span></p></td><td class="c16"><p class="c4"><span>7.9</span></p></td><td class="c16"><p class="c4"><span>13.1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.hedvpls048g0"></a><span>Digestion:<br></span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>18-3</span></p></td><td class="c13"><p class="c4"><span>11</span></p></td><td class="c13"><p class="c4"><span>11-16</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>7</span></p></td><td class="c13"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>enzyme1</span></p></td><td class="c5"><p class="c4"><span>E &nbsp;1</span></p></td><td class="c13"><p class="c4"><span>X &nbsp; 1</span></p></td><td class="c13"><p class="c4"><span>X &nbsp;1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>enzyme 2</span></p></td><td class="c5"><p class="c4"><span>S &nbsp;1</span></p></td><td class="c13"><p class="c4"><span>P &nbsp; &nbsp;1</span></p></td><td class="c13"><p class="c4"><span>P &nbsp; 1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>9</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>12</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.3yfownuunxbz"></a><span>Ligation:</span></h3><h3 class="c4"><a name="h.3yfownuunxbz"></a><span>18-3&#65306;3ul &nbsp;16:1ul &nbsp; 2&#65306;6ul &nbsp;T4&#65306;1ul &nbsp;Buffer&#65306;2ul &nbsp; H2O&#65306;7ul</span></h3><h3 class="c29 c4"><a name="h.3yfownuunxbz"></a></h3><h3 class="c4"><a name="h.3yfownuunxbz"></a><span>18-3&#65306;3ul &nbsp;11-16&#65306;4ul &nbsp;2&#65306;6ul &nbsp; T4&#65306;1ul &nbsp; Buffer&#65306;2ul &nbsp;ddH2O&#65306;4ul</span></h3><h1 class="c4"><a name="h.tmngci922iwu"></a><span>9.2 </span></h1><h3 class="c4"><a name="h.wkppfbc5lyza"></a><span>1&#12289;Presrvation species of leuB and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.wkppfbc5lyza"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><h3 class="c4"><a name="h.wkppfbc5lyza"></a><span>1</span></h3></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>3</span></p></td><td class="c13"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>292.5</span></p></td><td class="c5"><p class="c4"><span>21.5</span></p></td><td class="c13"><p class="c4"><span>223.5</span></p></td><td class="c13"><p class="c4"><span>32.0</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.bi0spzoaltti"></a><span>Digestion:</span></h1><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>leuB</span></p></td><td class="c9"><a href="#" name="id.d705116cf752"></a><p class="c4"><span>K(pSB1C3)</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoRI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>PstI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>14</span></p></td><td class="c9"><p class="c4"><span>8.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>7.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.9sfoto1zfgis"></a><span>Check by small gel:</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>recover&#65306;</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>2&#12289;pick single colony of 18-3&amp;11-16 and 18-3&amp;11 and culture in shaker</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>3&#12289;transform BBa_I751250,as LuxI_AHL producer&#12290;Transform BBa_J04450(placI+mRFP)</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>9.3</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>1&#12289;ligate leuB, K(pSB1C3) carrier and transform</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>Before Concentration</span></p></td><td class="c9"><p class="c4"><span>After &nbsp;Concentration</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>LeuB</span></p></td><td class="c9"><p class="c4"><span>15</span></p></td><td class="c9"><p class="c4"><span>36</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1C3</span></p></td><td class="c9"><p class="c4"><span>8</span></p></td><td class="c9"><p class="c4"><span>25</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.6ii9ncjvqxui"></a><span>ligation</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>LeuB</span></p></td><td class="c1"><p class="c4"><span>Psb1c3</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>7</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.im43403pxmes"></a><span>2&#12289;extract plasmid of 8-3&amp;11-16 and 18-3&amp;11, digest&#65292;and check by small gel</span></h3><h3 class="c4"><a name="h.im43403pxmes"></a><span>Density:</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c34"><p class="c4"><span>18-3&amp;11-161</span></p></td><td class="c34"><p class="c4"><span>18-3&amp;11-162</span></p></td><td class="c18"><p class="c4"><span>18-3&amp;11 1</span></p></td><td class="c18"><a href="#" name="id.ad39c9b90daa"></a><p class="c4"><span>18-3&amp;11 2</span></p></td><td class="c52"><p class="c4"><span>5-4&#65288;AC&#65289;1 </span></p></td><td class="c56"><p class="c4"><span>5-4&#65288;AC&#65289; 2</span></p></td><td class="c53"><p class="c4"><span>2-6-3</span></p></td></tr><tr class="c12"><td class="c34"><p class="c4"><span>265</span></p></td><td class="c34"><p class="c4"><span>242</span></p></td><td class="c18"><p class="c4"><span>192</span></p></td><td class="c18"><p class="c4"><span>217.5</span></p></td><td class="c52"><p class="c4"><span>193</span></p></td><td class="c56"><p class="c4"><span>72</span></p></td><td class="c53"><p class="c4"><span>63.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.qevj1yc7kkwj"></a><span>digestion&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11-16(1)</span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11-16(2)</span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11(1)</span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11(2)</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>3.5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>EcoRI</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>PstI</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>ddH2O</span></p></td><td class="c1"><p class="c4"><span>12.5</span></p></td><td class="c1"><p class="c4"><span>12</span></p></td><td class="c1"><p class="c4"><span>11</span></p></td><td class="c1"><p class="c4"><span>11.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.s7dxsac1fmjh"></a><span>Check by gel&#65306;</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.f5jqe09y4j66"></a><span>2&#12289;ligate 5-4&amp;2-6-3</span></h3><h3 class="c4"><a name="h.6o1u60ekkzdx"></a><span>ligation</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>K</span></p></td><td class="c0"><p class="c4"><span>5-4</span></p></td><td class="c0"><p class="c4"><span>2-6-3</span></p></td><td class="c0"><p class="c4"><span>T4&#36830;&#25509;&#37238;</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.h3yoy287vb0g"></a><span>9&#65294;4</span></h1><h3 class="c4"><a name="h.djojukljacfp"></a><span>1&#12289;transform 5-4&amp;2-6-3&#65292;extract plasmid of &nbsp;2-6-3&#19982;2</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>2&#12289;transform Pea Rhizobium</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>9.5</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>1&#12289;Presrvation species of leuB and extract plasmid,measure density, digest, PCR ,check by gel</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>PCR</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>ddH2O</span></p></td><td class="c0"><p class="c4"><span>37.5</span></p></td><td class="c0"><p class="c4"><span>37</span></p></td><td class="c0"><p class="c4"><span>36.5</span></p></td><td class="c6"><p class="c4"><span>36.5</span></p></td><td class="c6"><p class="c4"><span>36.5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>dNTPmix</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>VF2</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>VR</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>tamplate</span></p></td><td class="c0"><p class="c4"><span>0</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Taq</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c6"><p class="c4"><span>0.5</span></p></td><td class="c6"><p class="c4"><span>0.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.4vezb87sj3gs"></a><span>Check by gel </span></h3><p class="c4"><img height="527" src="images/image02.jpg" width="527"></p><h3 class="c4"><a name="h.72c4zg6pthgu"></a><span>digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>LeuB &nbsp;1</span></p></td><td class="c9"><p class="c4"><span>LeuB &nbsp;3</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BufferM</span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>1.1</span></p></td><td class="c9"><p class="c4"><span>1.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>SpeI</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>XbaI</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.prk8j67a3erf"></a><span>Check by gel </span></h3><h1 class="c4"><a name="h.ftq4x9fezl9p"></a><span>9.6</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.tzu0yabpanct"></a><span>1&#12289;Presrvation species of 54263 and extract plasmid, digest, PCR ,check by gel</span></h3><h3 class="c29 c4"><a name="h.tzu0yabpanct"></a></h3><h3 class="c4"><a name="h.3i6aolmrnp3z"></a><span>digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>1.4</span></p></td><td class="c13"><p class="c4"><span>1.4</span></p></td><td class="c13"><p class="c4"><span>1.7</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>0.5</span></p></td><td class="c13"><p class="c4"><span>0.5</span></p></td><td class="c13"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>2.5</span></p></td><td class="c13"><p class="c4"><span>2.5</span></p></td><td class="c13"><p class="c4"><span>2.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI</span></p></td><td class="c5"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td></tr></tbody></table><h1 class="c49 c4"><a name="h.woe6zwxsksvp"></a></h1><h1 class="c4"><a name="h.7wkxrcs7xpbv"></a><span>9.7</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.uxiz6xa9xofa"></a><span>1&#12289;measure OD of Deficient cell</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>MM&#65288;leuB-&#65289;</span></p></td><td class="c0"><p class="c4"><span>1 &nbsp;leuB+</span></p></td><td class="c0"><p class="c4"><span>2 &nbsp; leuB+</span></p></td><td class="c6"><p class="c4"><span>3 &nbsp; leuB+</span></p></td><td class="c6"><p class="c4"><span>4 &nbsp;leuB+</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>OD&#65288;A340&#65289;</span></p></td><td class="c0"><p class="c4"><span>1.611-1</span></p></td><td class="c0"><p class="c4"><span>1.698-1</span></p></td><td class="c0"><p class="c4"><span>1.818-1</span></p></td><td class="c6"><p class="c4"><span>1.548-1</span></p></td><td class="c6"><p class="c4"><span>1.881-1</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.w97sjfemswol"></a><span>2&#12289;digest &nbsp;leuB&#12289;&#9314;&#12289;K115001</span></h3><h3 class="c4"><a name="h.mfs7a181f07r"></a><span>digestion&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c26"><p class="c2"><span></span></p></td><td class="c30"><p class="c4"><span>8</span></p></td><td class="c25"><p class="c4"><span>6</span></p></td><td class="c46"><p class="c4"><span>LeuB</span></p></td><td class="c30"><p class="c4"><span>3</span></p></td><td class="c24"><p class="c4"><span>T1</span></p></td><td class="c26"><p class="c4"><span>T2</span></p></td><td class="c24"><p class="c4"><span>T3</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>DNA</span></p></td><td class="c30"><p class="c4"><span>9</span></p></td><td class="c25"><p class="c4"><span>9</span></p></td><td class="c46"><p class="c4"><span>10</span></p></td><td class="c30"><p class="c4"><span>10</span></p></td><td class="c24"><p class="c4"><span>10</span></p></td><td class="c26"><p class="c4"><span>13</span></p></td><td class="c24"><p class="c4"><span>12</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>Buffer</span></p></td><td class="c30"><p class="c4"><span>2</span></p></td><td class="c25"><p class="c4"><span>2</span></p></td><td class="c46"><p class="c4"><span>2</span></p></td><td class="c30"><p class="c4"><span>2</span></p></td><td class="c24"><p class="c4"><span>2</span></p></td><td class="c26"><p class="c4"><span>2</span></p></td><td class="c24"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>DdH2O</span></p></td><td class="c30"><p class="c4"><span>7</span></p></td><td class="c25"><p class="c4"><span>6.6</span></p></td><td class="c46"><p class="c4"><span>5.6</span></p></td><td class="c30"><p class="c4"><span>5.6</span></p></td><td class="c24"><p class="c4"><span>5.6</span></p></td><td class="c26"><p class="c4"><span>2.6</span></p></td><td class="c24"><p class="c4"><span>3.6</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>EcoRI</span></p></td><td class="c30"><p class="c4"><span>1</span></p></td><td class="c25"><p class="c4"><span>1.2</span></p></td><td class="c46"><p class="c4"><span>1.2</span></p></td><td class="c30"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td><td class="c26"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>SpeI</span></p></td><td class="c30"><p class="c4"><span>1</span></p></td><td class="c25"><p class="c4"><span>1.2</span></p></td><td class="c46"><p class="c4"><span>1.2</span></p></td><td class="c30"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td><td class="c26"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>density&#65306;</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>LeuB 98.0</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>T &nbsp;1:93.5 &nbsp; &nbsp; 2:73.5 &nbsp; &nbsp;3:87</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>3 &nbsp;1:97 &nbsp; &nbsp; &nbsp; 2:73 &nbsp; &nbsp; 3:82</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>9.8</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>1&#12289;extract plasmid of 6&#65292;and digest with LeuB</span></h3><h3 class="c4"><a name="h.9dqcvm6xpq2y"></a><span>Density of 6&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>57</span></p></td><td class="c9"><p class="c4"><span>68</span></p></td><td class="c9"><p class="c4"><span>50</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.ufa9zdoc4n5v"></a><span>digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>LeuB</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>10</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>9.6</span></p></td><td class="c13"><p class="c4"><span>9.6</span></p></td><td class="c13"><p class="c4"><span>5.6</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>SpeI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.tgspjxa7cy05"></a><span>2&#12289;ligate leuB&amp;3</span></h3><h3 class="c4"><a name="h.tgspjxa7cy05"></a><span>ligation&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c0"><p class="c4"><span>leuB</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c6"><p class="c4"><span>T carrier</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>0.7</span></p></td><td class="c6"><p class="c4"><span>10</span></p></td><td class="c6"><p class="c4"><span>3.3</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.2tn7lcrp7780"></a><span>9.9</span></h1><h3 class="c4"><a name="h.ix5rjouwfyue"></a><span>1&#12289;check 6, leuB, 8</span></h3><h3 class="c4"><a name="h.ix5rjouwfyue"></a><span>Gel&#65306;</span></h3><h3 class="c4"><a name="h.ix5rjouwfyue"></a><span>Ligate 8&amp;6&#12289;6&amp;leuB</span></h3><h3 class="c4"><a name="h.jizjvyn0z6vd"></a><span>Ligation of 8&amp;6&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>T carrier</span></p></td><td class="c0"><p class="c4"><span>8</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>10</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.sojo5w7w5q9c"></a><span>Ligation of &nbsp;6&amp;leuB</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>T carrier</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>LeuB</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>10</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.vnbfx386r4a2"></a><span>9.10</span></h1><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>1&#12289;transform 8&amp;6 and &nbsp;leuB</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>2&#12289;culture leuB&amp;3 in LB medium</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>3&#12289;device checking&#65306;</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>Device1 has strong Fluorescent and device 2 hasslight one</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>Picture:</span></h3><h1 class="c4 c49"><a name="h.1ummjyzat1wc"></a></h1><h1 class="c4"><a name="h.p1bniw9xi6lt"></a><span>9.11</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.j1mlghz0dpg9"></a><span>1&#12289;PCR&#65306;sinR&#12289;pro-RBS-sinR&#12289;raiR&#12289;raiR-pro</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.fcdrc1dlv06o"></a><span>density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c37"><p class="c4"><span>sinI</span></p></td><td class="c37"><p class="c4"><span>12.8</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>SinI pro</span></p></td><td class="c37"><p class="c4"><span>9.6</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pro-RBS-sinI</span></p></td><td class="c37"><p class="c4"><span>15.1</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>RaiI</span></p></td><td class="c37"><p class="c4"><span>14.2</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>RaiI pro</span></p></td><td class="c37"><p class="c4"><span>9.4</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pro-RBS-raiI</span></p></td><td class="c37"><p class="c4"><span>11.4</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>SinR pro</span></p></td><td class="c37"><p class="c4"><span>11.5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pro-RBS-raiR</span></p></td><td class="c37"><p class="c4"><span>15.0</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>2&#12289;pick single colony of 6&amp;leuB</span></h3><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>9.12</span></h3><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>1&#12289;digest 21&#12289;22 and ligate</span></h3><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>digestion</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>21</span></p></td><td class="c13"><p class="c4"><span>22</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>NDA</span></p></td><td class="c5"><p class="c4"><span>13.6</span></p></td><td class="c13"><p class="c4"><span>15</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>0.6</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>SpeI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.7q3di5col2wi"></a><span>ligation</span></h3><h3 class="c4"><a name="h.7q3di5col2wi"></a><span>21&amp;3</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>21</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>Buffer </span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td><td class="c6"><p class="c4"><span>T</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td><td class="c6"><p class="c4"><span>10</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.8g2gy677wg7m"></a><span>22&amp;3</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>22</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td><td class="c6"><p class="c4"><span>T</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>10</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.78fosknlspxo"></a><span>2&#12289;extract plasmid of leuB-6 and measure density</span></h3><h3 class="c4"><a name="h.78fosknlspxo"></a><span>density&#65306;16.5</span></h3><h1 class="c4"><a name="h.z2gmdjiy1hon"></a><span>9&#65294;13</span></h1><h3 class="c4"><a name="h.slxf8ucbmfw5"></a><span>1&#12289;transform and culture 21&amp;3,22&amp;3,8&amp;6&#65292;recover PSB1C3 carrier</span></h3><h3 class="c4"><a name="h.xnxdtzar9e2f"></a><span>2.Pcr</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>Pfu with MgSO4 buffer</span></p></td><td class="c21"><p class="c4"><span>dNTP</span></p></td><td class="c17"><p class="c4"><span>PF</span></p></td><td class="c21"><p class="c4"><span>PR</span></p></td><td class="c21"><p class="c4"><span>Template</span></p></td><td class="c21"><p class="c4"><span>Pfu</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>38</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c17"><p class="c4"><span>5</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>4</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c17"><p class="c4"><span>1</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>1</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>0.5</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>0.5</span><span class="c3">&micro;</span><span>l</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.vyykrtrhy0we"></a><span>Results&#65306;failed</span></h3><h3 class="c4"><a name="h.vyykrtrhy0we"></a><span>3.digest sinR, sinI,pro- sinI</span></h3><h3 class="c4"><a name="h.2kxssry4fo1d"></a><span>Digestion:20</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>sinR</span></p></td><td class="c13"><p class="c4"><span>sinI</span></p></td><td class="c13"><p class="c4"><span>pro- sinI</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>11</span></p></td><td class="c13"><p class="c4"><span>16</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>H</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>ddH2O</span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>5</span></p></td><td class="c13"><p class="c4"><span>0</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.3n89uor9jv7a"></a><span>9.15</span></h1><h3 class="c4"><a name="h.mwd71ak11zm"></a><span>Digestion of Leu&amp;3 :20</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c4"><span>number</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>9</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>H(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>7</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.s3eqhw38nr24"></a><span>9.16</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.8um0wy4nrt8u"></a><span>Digest 6 and leub</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>Leu</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c9"><p class="c4"><span>15</span></p></td><td class="c9"><p class="c4"><span>9</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>M(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>7</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzymew</span></p></td><td class="c9"><p class="c4"><span class="c7">Spe</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l) </span><span class="c3">1</span></p></td><td class="c9"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l) 1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l) 1</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.5jlkua87etap"></a><span>9.18</span></h1><h3 class="c4"><a name="h.wll2rnwql4h5"></a><span>Ligate Leu and 6 using 3A</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Leu</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>buffer</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>10</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">2&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">6&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">2&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">1&micro;</span><span>l</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.8d09k2ryw264"></a><span>Gel picture&#65306;</span></h3><p class="c4"><img height="273" src="images/image03.jpg" width="562"></p><h3 class="c4"><a name="h.pzff1yr1mq6n"></a><span>Digest 6</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>number</span></p></td><td class="c1"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>&nbsp;(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>9</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>8</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.rjhja6hena4r"></a><span>9.20</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.doclcz4jf63n"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c38"><p class="c4"><span>number</span></p></td><td class="c50"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c50"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>6</span></p></td><td class="c50"><p class="c4"><span>179.5</span></p></td><td class="c50"><p class="c4"><span>1.851</span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>pSB1C3-1</span></p></td><td class="c50"><p class="c4"><span>6.3</span></p></td><td class="c50"><p class="c2"><span></span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>pSB1C3-2</span></p></td><td class="c50"><p class="c4"><span>8.6</span></p></td><td class="c50"><p class="c4"><span>3.510</span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>pSB1C3-3</span></p></td><td class="c50"><p class="c4"><span>5.1</span></p></td><td class="c50"><p class="c2"><span></span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.xbnykbw91qca"></a><span>Digest Leu</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>M(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c19 c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>8</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ebm9jwpwvzye"></a><span>Ligate Leu&amp; 6</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>Leu</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>buffer</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>ddH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>7</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.gx5ptw3fqhxn"></a><span>9.21</span></h1><h3 class="c4"><a name="h.t4pz3r81wrf0"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-3</span></p></td><td class="c9"><p class="c4"><span>29.5</span></p></td><td class="c9"><p class="c4"><span>1.666</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-2</span></p></td><td class="c9"><p class="c4"><span>28.5</span></p></td><td class="c9"><p class="c4"><span>1.885</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-1</span></p></td><td class="c9"><p class="c4"><span>23.0</span></p></td><td class="c9"><p class="c4"><span>1.818</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C0076</span></p></td><td class="c9"><p class="c4"><span>161.0</span></p></td><td class="c9"><p class="c4"><span>1.793</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>E0020e0fp-1</span></p></td><td class="c9"><p class="c4"><span>28.5</span></p></td><td class="c9"><p class="c4"><span>1.676</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>E0020e0fp-2</span></p></td><td class="c9"><p class="c4"><span>19.5</span></p></td><td class="c9"><p class="c4"><span>2.19