Blackburn:Yeast Colony PCR
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Revision as of 00:22, 12 June 2006
This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.
- Standard PCR machine, tubes
- Qiagen Taq Polymerase Kit with Q-solution
- A small yeast colony
- 0.02M NaOH (3uL per reaction)
Yeast Cell Lysis
- Aliquot 3uL 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.
- [optional] Quick-freeze the cells in liquid nitrogen
- I normally skip this step. Works just fine.
- 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 sometime. Keep on ice or freeze for longer.
- Prepare the master mix solution containing:
- 5uL 5X Q-solution
- 2.5uL 10X PCR Buffer
- 0.5uL dNTPs (10mM each)
- 0.5uL foward primer (100uM)
- 0.5uL reverse primer (100uM)
- 0.25uL Taq
- 12.75uL ddH2O
- Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume).
- Run the following PCR cycle:
- 5 min at 94C
- 30 cycles of:
- 30 sec at 94C
- 30 sec at 55C (or appropriate annealing temperature)
- 1 min/kbp at 72C
- 10 min at 72C
- 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.