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 of bacteria E. coli DH5α	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	x °C	30 s - 1 min	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.

Team:Paris Saclay/protocols/pcr