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Team:Paris Saclay/protocols/pcr
From 2013.igem.org
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Revision as of 16:11, 27 September 2013
PCR for the genomic DNA of bacteria
1. In a cold PCR tube (Eppendorf), pipet in the presented order the solutions listed in Table 1.
| component | volume/50µL reaction | volume/20µL reaction |
|---|---|---|
|
H2O |
Add to 50 μl |
Add to 20 μl |
|
5x Phusion HF Buffer High-fidelity |
10 μl |
4 μl |
|
10 mM dNTPs |
1 μl |
0.4 μl |
|
Primer A |
x μl |
x μl |
|
Primer B |
x μl |
x μl |
|
Template DNA: Genomic DNA |
x μl |
x μl |
|
Phusion DNA polymerasse |
0.5 μl |
0.2 μl
|
2. Program the PCR machine according to the Table 2.
| Cycle step | temperature | time | cycle |
|---|---|---|---|
|
Initial denaturation |
95 - 98 °C |
30 s – 3 min |
1 |
|
Denaturation |
95 - 98 °C |
10 – 30 s |
25-30 |
|
Annealing |
variable |
30 s |
25-30 |
|
Extension |
72°C |
30 s – 2 min |
25-30 |
|
Final extension |
72°C |
10min |
1 |
|
Final extension |
4-8°C |
hold |
1 |
3. Determine the number of oligo by electrophoresis.
