Blackburn:Yeast Colony PCR v2.0: Difference between revisions
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(New page: ==Overview== This is a quick and easy yeast colony PCR protocol that does not require zymolyase step. ==Materials== *Standard PCR machine, tubes *[http://www.qiagen.com/Products/Pcr/Taq...) |
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#Transfer 1uL of boiled samples to the master mix aliquots (a multi-channel pipette is helpful here). | #Transfer 1uL of boiled samples to the master mix aliquots (a multi-channel pipette is helpful here). | ||
#Run the following PCR cycle: | #Run the following PCR cycle: | ||
##5 min at | ##5 min at 95C | ||
##30 cycles of: | ##30 cycles of: | ||
### | ###5 sec at 95C | ||
### | ###10 sec at 55C (or appropriate annealing temperature) | ||
###1 min/kbp at 72C | ###1 min/kbp at 72C | ||
##10 min at 72C | ##10 min at 72C |
Revision as of 15:55, 20 March 2009
Overview
This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.
Materials
- 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.
Procedure
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.
PCR
- 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:
- 5 sec at 95C
- 10 sec at 55C (or appropriate annealing temperature)
- 1 min/kbp at 72C
- 10 min at 72C
Notes
- 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.