Wittrup: Yeast colony PCR
Revision as of 07:23, 24 April 2007 by Ssazinsk (New page: ==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 forwar...)
Yeast colony PCR to amplify genomic or plasmid DNA
- 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
- Prepare the PCR mix, as follows (for 1x):
- 2.0 μL yeast [add last: see Steps 3 and 4]
- 2.0 μL 10x PCR buffer
- 0.6 μL 50 mM MgCl2
- 0.4 μL 10 mM dNTPs
- 1.0 μL Forward primer, 10 uM
- 1.0 μL Reverse primer, 10 uM
- 0.5 μL Taq (5 U/ul)
- 12.5 μL ddH2O
- 20.0 μL total reaction volume
- Pick a yeast colony from a plate into 20 ul of ddH2O in a microcentrifuge tube.
- 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.
- Microwave the yeast for 30 seconds.
- Add 2 μL of the yeast template to the PCR mix.
- Cycling conditions:
- Cycle 1: 4 min at 95 °C
- Cycle 2-35: [1 min at 95 °C, 1 min at 55 °C, 1 min at 72 °C]
- Last: 10 min at 72°C
- The microwaving step shouldn't be necessary, although I've found that it gives better yields (SLS).
- You can substitute commercial 10x PCR buffers and MgCl2 for the buffers listed above (SLS).
Relevant papers and books
- Adapted from “Molecular Cloning”, 3rd edition, by Sanbrook & Russell, Cold Spring Harbor Laboratory Press, 2001. “Analyzing Yeast Colonies by PCR” [www.cshprotocols.org]