Wittrup: Yeast colony PCR

Overview
Yeast colony PCR to amplify genomic or plasmid DNA

Materials

 * PCR mix
 * 10x PCR buffer [0.125 M Tris-Cl (pH 8.5), 0.56 M KCl]
 * 50 mM MgCl2
 * 10 mM dNTPs
 * 10 uM forward PCR primer
 * 10 uM reverse PCR primer
 * Taq polymerase

Procedure

 * 1) Prepare the PCR mix, as follows (for 1x):
 * 2) *2.0 &mu;L yeast [add last: see Steps 3 and 4]
 * 3) *2.0 &mu;L 10x PCR buffer
 * 4) *0.6 &mu;L 50 mM MgCl2
 * 5) *0.4 &mu;L 10 mM dNTPs
 * 6) *1.0 &mu;L Forward primer, 10 uM
 * 7) *1.0 &mu;L Reverse primer, 10 uM
 * 8) *0.5 &mu;L Taq (5 U/ul)
 * 9) *12.5 &mu;L ddH2O
 * 10) *20.0 &mu;L total reaction volume
 * 11) Pick a yeast colony from a plate into 20 ul of ddH2O in a microcentrifuge tube.
 * 12) *It’s important not to take too much yeast and/or agar – it doesn’t take much yeast for the PCR to work. Use a yellow tip (200 ul) pipet tip to gently dab the colony and transfer to the tube of water.
 * 13) Microwave the yeast for 30 seconds.
 * 14) Add 2 &mu;L of the yeast template to the PCR mix.
 * 15) Cycling conditions:
 * 16) *Cycle 1: 4 min at 95 °C
 * 17) *Cycle 2-35: [1 min at 95 °C, 1 min at 55 °C, 1 min at 72 °C]
 * 18) *Last: 10 min at 72°C