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 new version of the protocol uses 10uL PCR reactions, significantly reducing the reagent costs.
Older version: Blackburn Lab: Quick and Easy Yeast Colony PCR
- 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 criticial.
- A small yeast colony
- 0.02M NaOH (10uL per reaction)
- Multi-channel pipettes are very helpful.
Yeast Cell Lysis
- Aliquot 10uL of 0.02M NaOH into PCR tubes.
- 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.
- 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.
- 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
- Aliquot 9uL of the master mix solution into fresh PCR tubes.
- Transfer 1uL of boiled samples to the master mix aliquots (a multi-channel pipette is helpful here).
- Run the following PCR cycle:
- 5 min at 95C
- 30 cycles of:
- 10 sec at 95C
- 10 sec at 50C (or appropriate annealing temperature)
- 1 min/kbp at 72C (I generally do 30 sec)
- 10 min at 72C (I don't think this step is critical)
- 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.
Following is the Blackburn:Yeast Colony PCR v2.0 protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi