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== | ||
* | *Standard PCR machine, tubes | ||
* | *[http://www.qiagen.com/Products/Pcr/TaqSystem/TaqDnaPolymerase.aspx Qiagen Taq Polymerase Kit with Q-solution] | ||
* | *A small yeast colony | ||
* | *0.02M NaOH (3uL per reaction) | ||
==Procedure== | ==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. | |||
==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.