Template:IONIS Paris/Notebook/01-07-15

PCR pDawn BioBricks

PCR pDawn III

Aim: first step for pDawn amplification and mutagenesis
Mix PCR preparation:
pDawn III
MQ water 39,5 µL
Mg2+ 50 mM 1,5 µL
Buffer RB 5 µL
dNTPs 10µL 1 µL
Primer Fwd 50µM 1µL pDawn Fwd III
Primer Rev 50µM 1µL pDawn Rev III
DNA 1µL pDawn
Taq Pol 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

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 totale
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

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

Soft band at 1500 bp, good but not enough


1 July 15