Wittrup: Yeast colony PCR

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Contents

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):
    • 2.0 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
  2. 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.
  3. Microwave the yeast for 30 seconds.
  4. Add 2 μL of the yeast template to the PCR mix.
  5. 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


Notes

  1. The microwaving step shouldn't be necessary, although I've found that it gives better yields (SLS).
  2. You can substitute commercial 10x PCR buffers and MgCl2 for the buffers listed above (SLS).


References

Relevant papers and books

  1. Adapted from “Molecular Cloning”, 3rd edition, by Sanbrook & Russell, Cold Spring Harbor Laboratory Press, 2001. “Analyzing Yeast Colonies by PCR” [www.cshprotocols.org]
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