Blackburn:Yeast Colony PCR: Difference between revisions
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Updated Protocol: [[Blackburn:Yeast_Colony_PCR_v2.0|Blackburn Lab: Quick and Easy Yeast Colony PCR v2.0]] | |||
==Overview== | ==Overview== | ||
This is a quick and easy yeast colony PCR protocol that does not require zymolyase step. | This is a quick and easy yeast colony PCR protocol that does not require zymolyase step. | ||
Updated Protocol: [[Blackburn:Yeast_Colony_PCR_v2.0|Blackburn Lab: Quick and Easy Yeast Colony PCR v2.0]] | |||
==Materials== | ==Materials== | ||
| Line 20: | Line 24: | ||
#Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes. | #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. | #*In the mean time, prepare the master mix for the PCR reaction. | ||
#*The boiled samples are stable at room temp for | #*The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer. | ||
===PCR=== | ===PCR=== | ||
| Line 42: | Line 46: | ||
==Notes== | ==Notes== | ||
*Note that we use 0.5uL of 100uM primer, which is about ten-times more than standard PCR protocols. | |||
*The PCR product can be loaded onto agarose gels directly without addition of loading buffer. | *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. | *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. | |||
==Contact== | ==Contact== | ||
[[Special:Emailuser/Tetm2002|Tet]] ([http://biochemistry.ucsf.edu/~blackburn/ Blackburn Lab]) | |||
Latest revision as of 18:13, 20 March 2009
Updated Protocol: Blackburn Lab: Quick and Easy Yeast Colony PCR v2.0
Overview
This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.
Updated Protocol: Blackburn Lab: Quick and Easy Yeast Colony PCR v2.0
Materials
- Standard PCR machine, tubes
- Qiagen Taq Polymerase Kit with Q-solution
- A small yeast colony
- 0.02M NaOH (3uL per reaction)
Procedure
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 some time. Keep on ice or freeze for longer.
PCR
- 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
Notes
- Note that we use 0.5uL of 100uM primer, which 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.
