Team:OUC-China/Result/date

From 2011.igem.org

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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

1Taq 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

Xba(µl)

1

1

1

Pst(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

Spe(µl) 1

Xba(µl) 1

Pst(µl) 1

9.18

Ligate Leu and 6 using 3A

Leu

6

buffer

T4

10µl

l

l

l

l

Gel picture:

Digest 6

number

DNA(µl)

Buffer 10× (µl)

Pst(µl)

ddH2O(µl)

6

9

2

1

8

9.20

Measure density

number

ng/µl

A260A280

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)

Pst(µl)

Xba(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

A260A280

raipro-3

29.5

1.666

raipro-2

28.5

1.885

raipro-1

23.0

1.818

C0076

161.0

1.793

E0020e0fp-1

28.5

1.676

E0020e0fp-2

19.5

2.19