Colony PCR from bacteria
1.
Add 100ul sterile milliQ water to either 0.5ml eppendorfs or a semi-skirted 96-well PCR
plate. (DO NOT THROW THIS AWAY – store in fridge).
2.
Use a pipette tip to pick a single colony and mix with the water.
3.
Close tube or cover plate with foil seal and vortex or shake briefly.
4.
Make up the following master mix (this mix is for 20 reactions):
5.
20ul
10x PCR buffer without MgCl2
164ul
Sterile milliQ water
6ul
50mM MgCl2
4ul
10mM dNTPs
2ul
Forward primer (100uM)
2ul
Reverse primer (100uM)
1ul
Taq DNA polymerase
To each well in a semi-skirted 96-well PCR plate add 9ul master mix plus 1ul of the
colony/water suspension.
6.
PCR programme:
94oC
30 seconds
50oC
1 minute
72oC
1 min per kb
72oC
7 minutes
4o C
10 minutes
30 cycles
7.
Add 5ul loading buffer to each reaction.
8.
Run on a 1% gel at 100V for approx. 45 minutes.