Template:IONIS Paris/Notebook/27-07-15
PCR pDawn: whole fragment
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 |
T°C | Time | Cycle | |
---|---|---|---|
Initial denaturation | |||
Denaturation | |||
Annealing | |||
Extension | |||
Final extension | |||
Hold |
No amplification
Gibson Assembly
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 | |
0.000000031155 | 0.000000031155 | |||||||
VVD1 | |
0.000000096698 | 0.000000031155 | |||||||
VVD2 | |
0.000000047103 | 0.000000031155 | |||||||
YN155 I | |
0.000000064805 | 0.000000031155 | |||||||
YN155 II | |
0.000000114117 | 0.000000031155 |
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