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From 2011.igem.org

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	2.191

Digestion 5µl

number	raiⅠpro-1	raiⅠpro-2	raiⅠpro-3
DNA(µl)	4	4	4
Buffer 10×H (µl)	0.5	0.5	0.5
EcoRI(µl)	0.25	0.25	0.25
PstI(µl)	0.25	0.25	0.25

20µl

number	DNA(µl)	Buffer 10×M(µl)	ddH2O(µl)	PstⅠ(µl)	XbaⅠ(µl)
C0076	6.5	2	9.5	1	1

50µl

number	DNA(µl)	Buffer 10×M(µl)	ddH2O(µl)	PstⅠ(µl)	XbaⅠ(µl)
E0020	24	5	18	1.5	1.5

9.23

devive	一		二		三
number	5-4(AC)	Leu-6(A)	18-3-11(AK)	Leu-6(A)	Leu-6(A)	13(A)
DNA(µl)	16	13.5	6.5	13..5	13.5	12
Buffer (µl)	2	2	2	2	2	2
ddH2O(µl)	0	2.5	9.5	2.5	2.5	4
enzyme	EcoRI1	XbaⅠ1	EcoRI 1	XbaⅠ1	EcoRI 1	XbaⅠ1
	SpeⅠ1	PstⅠ1	SpeⅠ1	PstⅠ1	SpeⅠ1	PstⅠ1

Measure density

number	ng/µl	A260∕A280
C0076	21	2.154
Clue-6	72.5	1.933
Ce0020	22	2.064
6	82	2.130
13	82	1.864
2	45	1.738

9.24

First ligation

number	1		2		3
part	18-3-11	2.5	13	2	C0076	5
	Leu-6	2	Leu-6	2	6	3
cariier	AC 3.5		AC 3.5
buffer	2		2		2
T4	1		1		1
ddH2O	9		9.5		9

9.25

Second ligation

number	1		2
part	E0020	9	5-4	4
	6	9	Leu-6	2.5
cariier			AK 1.5
buffer	2		2
T4	1.5		1
ddH2O	5.5		9

Measure density

number		ng/µl	A260∕A280
3	1	29	2.041
	2	38	2.171
	3	35	2.265
E0020	1	69	1.816
	2	31	1.956
	3	45	1.957
5-4	1	110.5	1.939
	2	152.0	1.900
	3	76.5	1.987

9.26

Measure density

number	ng/µl	A260∕A280
14-19	112.5	1.875
RFP-1	145.0	1.859
RFP-2	138.5	1.884
RFP-3	162.0	1.873

9.27

Measure density

number	ng/µl	A260∕A280
C1	18.0	2.022
C2	26.0	2.097
C3	25.0	2.041
C4	23.0	1.862
rhiR	18.5	1.900
leuB	12.1	2.000
14-119-13	9.6
RFP	12.4	2.041

9.30

1. measure the density of 3 and digest

Density  (1) 78 ng/ul   (2)62ng/ul

Digestion

DNA	Buffer	XbaI	PstI
16	2	1	1

2.measure density of 14&19

(1)112ng/ul   (2)84ng/ul

10.1

1.measure density

3.2	3.3	C0076&6	C0076&6
273.5	275	164	169