"

From 2014.igem.org

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	Initial concentration	Final concentration	Example for 50μl
H2O	-	Add to final volume	37.75μl (50μl final)
5x Phusion HF Buffer High-fidelity	10X	1X	5μl
dNTPs	10mM	200μM	1μl
Primer A	100μM	0.5μM	2.5μl
Primer B	100μM	0.5μM	2.5μl
Template DNA: Genomic DNA	-	1μl	1μl
Phusion DNA polymerasse	-	0.25μl	0.25μl

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. Verify correct amplification by agarose gel electrophoresis.