Template:IONIS Paris/Notebook/06-07-15

PCR pDawn BioBricks

PCR pDawn III (3)

Aim: first step for pDawn amplification and mutagenesis
Mix PCR preparation:
pDawn III pDawn III
MQ water 25,5 µL 24 µL
Mg2+ 50mM / 1,5 µL
Q5 Buffer 10 µL 10 µL
High GC content Buffer 10 µL 10 µL
dNTPs 10µM 1 µL 1 µL
Primer Fwd 50µM 1µL pDawn Fwd III 1µL pDawn Fwd III
Primer Rev 50µM 1µL pDawn Rev III 1µL pDawn Rev III
DNA 1µL pDawn 1µL pDawn
Taq Pol 0.5 µL 0.5 µL
PCR cycle – IGEM TAQ 2
T°C Time Cycle
Initial denaturation
95
30 sec
1
Denaturation
95
30 sec
17
Annealing
60
30 sec
Extension
72
2 min
Final extension
72
5 min
1
Hold
4
Infinite

Electrophoresis


Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

Expected results

Expected results

Results

Results

No amplification

Gibson Assembly

Aim: first step of our VVD Biobrick
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 DNA Volume Volume Water Volume total
pSB1C3
91.11
2 200
1 430 000
0.000000063712
7.75
1.35
0.000000047103
3.50
1.50
5
VVD1
15.71
250
162 500
0.000000096698
5.10
2.05
0.000000047103
1.00
1.00
2
VVD2
7.65
250
162 500
0.000000047103
10.48
1.00
0.000000047103
1.00
0
1
YC155
128.31
400
260 000
0.000000493488
1.00
10.48
0.000000047103
1.00
9.00
10

DNA mix:

  • YC155: 1µL + 9µL of MQ water
  • VVD1: 1µL + 1µL of MQ water
  • VVD2: no dilution
  • pSB1C3: 3.5µL + 1.5µL of MQ water
Mix = 2µL of each
5µl used for the Gibson assembly added to 15µl of master mix

E.coli transformation


3 tubes: - 20µL of competent cells + 5µL of Gibson product - Idem - 20µL of competent cells as negative control
Same protocol as used before but only 200µl of LB has been added to resuspend bacteria. Then the final volume of bacteria which has been plated was 50 µl. 37°C, O/N


6 July 15