Template:IONIS Paris/Notebook/27-07-15

PCR pDawn: whole fragment

Mix PCR preparation:
pDawn pDawn
MQ water 35,5 µL 35,5 µL
Q5 Buffer 10 µL 10 µL
High GC content Buffer / 10 µL
dNTPs 10µM 1 µL 1 µL
Primer Fwd 50µM 1µL pDawn Fwd I 1µL pDawn Fwd I
Primer Rev 50µM 1µL pDawn Rev III-VI 1µL pDawn Rev III-VI
DNA 1µL pDawn 1µL pDawn
Taq Pol 0.5 µL 0.5 µL
PCR cycle – IGEM TAQ
T°C Time Cycle
Initial denaturation
98
5 min
1
Denaturation
98
30 sec
17
Annealing
60
30 sec
Extension
72
2 min
Final extension
72
10 min
1
Hold
4
Infinite

No amplification

Gibson Assembly

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
44.55
2 200
1 430 000
0.000000031155
3.66
1.00
0.000000031155
1.00
0.00
1.00
VVD1
15.71
250
162 500
0.000000096698
1.18
3.10
0.000000031155
1.00
2.10
3.10
VVD2
7.65
250
162 500
0.000000047103
2.42
1.51
0.000000031155
1.00
0.51
1.51
YN155 I
8.42
200
130 000
0.000000064805
1.76
2.08
0.000000031155
1.20
1.30
2.50
YN155 II
14.84
200
130 000
0.000000114117
1.00
3.66
0.000000031155
1.20
3.20
4.40

DNA mix:
YN155 1: 1,2µl + 1,3µl of MQ water
YN155 2: 1,2µl + 3,2µl of MQ water
VVD1 : 1µl + 2,1µl of MQ water
VVD2 : 1µl + 0,5 µl of MQ water
pSB1C3 : no dilution
mix= 2µl of each

5µl used for the Gibson assembly added to 15µl of master mix
Transformation of 100 µl of competent cells using 5 µl of Gibson product


27 July 15