Colony PCR is a nice way of screening colonies if you have to do a lot of them. It only really works with Taq. You wouldn’t do this unless you needed to screen many colonies, so you’ll want to do a mastermix of the water, buffer, oligos, and polymerase and split it into multiple 20uL reactions. You should modify the extension time for the size of your predicted PCR product, assume 45 sec per kb.
Set up the following reaction in a PCR tube:
15uL ddH2O 2uL 10x Taq or thermopol buffer 2uL dNTPs (2mM in each) 0.4uL ca998 (10uM) 0.4uL G00101 (10uM) 0.2uL Taq
To set this up, make and distribute your mastermix, then using a tip or toothpick stab a colony. Swoosh it around in the PCR reaction and then either streak an X on a plate, or drop it into a culture. If you don’t make a replica of the culture, your cells are gone!
The pcr program should be in the iGEM folder on the biorad machine.
Note: This protocol came from Chris's "PCR in practice" word doc. under 'files' in the google group.