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	15.4 μl
Taq PCR Buffer 10X	2 μl
dNTPs (10 mM each)	0.8 μl
MgCl2 (50mM)	0.8 μl
Primer Fwd (10 μM)	0.4 μl
Primer Rev (10 μM)	0.4 μl
Taq polymerase (5U/μl)	0.2 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
1X	72°C	7 min
	7°C	Inf