IGEM:Paris Bettencourt 2012/Protocols/PCR Jake

Extraction of Genomic DNA

1) Pick a single colony into 50 ul of H20.

Fresh colonies (grown that day) work best, but they can also come from 4 C.

2) Boil for 5 minutes.

1 ul of this can be used directly for PCR.

Best if used directly, but can also be stored at 4 C for a few days.

PCR Recipe (NEB Phusion Polymerase)

We use the Phusion polymerase because it has a low mutation rate. It also has a very fast extension time, which is useful for amplifying large constructs.

Reagent	Volume
	1x
Nuclease-free water	37 ul
5x Phusion HF Buffer	10 ul
10 mM dNTPs	1 ul
Forward Primer (10 uM)	0.5 ul
Reverse Primer (10 uM)	0.5 ul
Genomic DNA	1 ul
Phusion DNA Polymerase	0.5 ul ul
Total Volume	50 ul

Thermocycler Protocol: NEB Phusion
	Temp		Time
Start	98 C		30 sec	Melt
Cycle 1	98 C		5 sec	Melt	35 Cycles
Cycle 2	55 C		25 sec	Anneal
Cycle 3	72 C		30 sec per kb	Extend
Finish	72 C		5 min	Extend
Store	10 C		Forever	Store