Template:IONIS Paris/Notebook/04-06-15

Plasmid digestion (pSB1C3)

Aim: Extraction of the biobrick’s backbone for Gibson Assembly
Into aliquots, added plasmids or nothing (control) as following:
Tube pSBC3
Buffer 2.1
2 µL
Plasmid pSB1C3
17 µL
Enzyme EcoRI
0.5 µL
Enzyme PstI
0.5 µL

NEB double digestion Buffer 2.1:

  • EcoRI: 100%
  • PstI: 75%

37°C, 1h10
Storage: -20°C

Electrophoresis

Aim: electrophoresis for gel purification

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

Expected results

Expected results

Results

Results

We get expected bands
Extraction of the heaviest band of digested pSB1C3 and all the others expected bands for VVD 1, VVD 2, YC155, YN155 1, YN155 2.

Gel Purification

QIAquick gel extraction
VVD 1
180 mg
VVD 2
590 mg
YN155 1
180 mg
YN155 2
200 mg
YC155
120 mg
pSB1C3
190 mg

Concentrate into 30 µl of Elution Buffer Concentrate into 30 µl of Elution Buffer

Electrophoresis for quantification

Aim: quantification of ADN to perform Gibson Assembly

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

Expected results

Expected results

Results

Results

There is not enough DNA to detect and quantify pSB1C3 and YC155 Try again

Electrophoresis for quantification - 2nd

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

Expected results

Expected results

Results

Results

Quantification of DNA fragments by Image J:
Fragments Concentration (ng.µL-1) Lenght Molecular weight (g.mol-1) Molarity (mol.L-1) 1/ratio Dilution factor Molarity after dilution
pSB1C3
0.92
2 200
1 430 000
0.000000000642
177.7
1
0.000000000642
VVD1
15.71
250
162 500
0.000000096698
1.18
151
0.000000000642
VVD2
7.65
250
162 500
0.000000047103
2.42
73
0.000000000642
YC155
2.50
400
260 000
0.000000009631
11.85
15
0.000000000642
YN155.1
8.42
200
130 000
0.000000064805
1.76
101
0.000000000642
YN155.2
14.84
200
130 000
0.000000114117
1
178
0.000000000642

Liquid culture

Double click tube: 3 mL of LB broth + 3 µL of antibiotic
Plasmid
Antibiotic
Plasmid Antibiotic
Terminator T7
Cam
bFos
Amp
RBS
Cam
bJun
Amp
Control Negative
Cam
Control Negative
Amp

Concentrate into 30 µl of Elution Buffer Concentrate into 30 µl of Elution Buffer


4 June 15