Blackburn:Yeast Colony PCR v2.0

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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

• Standard PCR machine, tubes
• Qiagen Taq Polymerase Kit with Q-solution
  • Home-made Taq and buffer have been used successfully with this protocol, but the addition of the Q-solution, which is mainly betaine, is crticial.
• A small yeast colony
• 0.02M NaOH (10uL per reaction)
• Multi-channel pipettes are very helpful.

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.
   • I have been told adding too much yeast can inhibit the reaction.
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 (I don't think this step is critical)

Notes

• Q-solution is critical for this protocol; the main ingredient is 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 size 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)