Blackburn:Yeast Colony PCR v2.0
|Line 1:||Line 1:|
Revision as of 21:05, 20 March 2009
Back to Protocols
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
- 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.
- 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
- Note that the primer concentration we use is about ten-times more than standard PCR protocols.
- 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.