E coli colony PCR

This is a protocol to do PCR reactions on E. coli cells, directly after a transformation. (See here for colony PCR for yeast cells)

• Prepare mastermix

• Divide 19μl/sample over PCR reaction tubes/wells
• Prepare cells in water
• Add cell mixture to the mix

Re-suspend cells for each colony in 50μl DNAse free water and then add 1μl of this mix to the reaction.

Do sequencing reaction:
H2O	11.54 μl
Taq PCR Buffer 10X	1.5 μl
dNTPs (10 mM each)	0.6 μl
MgCl2 (50mM)	0.6 μl
Primer Fwd (10 μM)	0.3 μl
Primer Rev (10 μM)	0.3 μl
Taq polymerase (5U/μl)	0.16 μl

Run using program (bart -> colony PCR or cPCR)

Number of cycles	Temperature	Duration
1 X	94°C	2 min
35X	94°C	30 sec
	52°C	30 sec
	72°C	1 min 30 sec (or ~1kb/min)
1X	72°C	7 min
	7°C	Inf