Blackburn:Yeast Colony PCR v2.0

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This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.
This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.
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This new version of the protocol uses 10uL PCR reactions, significantly reducing the reagent costs.
This new version of the protocol uses 10uL PCR reactions, significantly reducing the reagent costs.

Revision as of 21:22, 20 March 2009

Back to Yeast Colony PCR

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Contents

Overview

This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.

This new version of the protocol uses 10uL PCR reactions, significantly reducing the reagent costs.

Older version: Blackburn Lab: Quick and Easy Yeast Colony PCR

Materials

Procedure

Yeast Cell Lysis

  1. Aliquot 10uL of 0.02M NaOH into PCR tubes.
  2. Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
    • If the solution is cloudy, you've added enough cells.
  3. Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes.
    • In the mean time, prepare the master mix for the PCR reaction.
    • The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.

PCR

  1. Prepare the master mix solution containing:
    • 2uL 5X Q-solution
    • 1uL 10X PCR Buffer
    • 0.2uL dNTPs (10mM each)
    • 0.2uL foward primer (100uM)
    • 0.2uL reverse primer (100uM)
    • 0.1uL Taq
    • 5.3uL ddH2O
  2. Aliquot 9uL of the master mix solution into fresh PCR tubes.
  3. Transfer 1uL of boiled samples to the master mix aliquots (a multi-channel pipette is helpful here).
  4. Run the following PCR cycle:
    1. 5 min at 95C
    2. 30 cycles of:
      1. 10 sec at 95C
      2. 10 sec at 50C (or appropriate annealing temperature)
      3. 1 min/kbp at 72C (I generally do 30 sec)
    3. 10 min at 72C

Notes

  • Q-solution is critical for this protocol; the main ingredient in betaine.
  • Note that the primer concentration we use is about ten-times more than standard PCR protocols.
  • The amount of taq used in this updated protocol is more per volume than the original protocol, but still less overall.
  • The PCR product can be loaded onto agarose gels directly without addition of loading buffer.
  • For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.
  • The expected PCR product should be as short as possible. Anything less than 1kbp can be easily amplified.
  • Generally, 2 distinct PCR products can be amplified in a single reaction. I do this to check the 5' and 3' ends of an integration in a single reaction (4 primers and different expected product sizes). This fails in about 5% of primer sets.

Contact

Tet (Blackburn Lab)

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