Blackburn:Yeast Colony PCR v2.0

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Line 48: Line 48:
###10 sec at 50C (or appropriate annealing temperature)
###10 sec at 50C (or appropriate annealing temperature)
###1 min/kbp at 72C (I generally do 30 sec)
###1 min/kbp at 72C (I generally do 30 sec)
-
##10 min at 72C
+
##10 min at 72C (I don't think this step is critical)
==Notes==
==Notes==
-
*Q-solution is critical for this protocol; the main ingredient in betaine.
+
*Q-solution is critical for this protocol; the main ingredient is betaine.
*Note that the primer concentration we use is about ten-times more than standard PCR protocols.
*Note that the primer concentration we use is about ten-times more than standard PCR protocols.
*The amount of taq used in this updated protocol is more per volume than the original protocol, but still less overall.
*The amount of taq used in this updated protocol is more per volume than the original protocol, but still less overall.
-
 
*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.
+
*The expected PCR product size should be as short as possible.  Anything less than 1kbp can be easily amplified.
*Generally, 2 distinct PCR products can be amplified in a single reaction.  I do this to check the 5' and 3' ends of an integration in a single reaction (4 primers and different expected product sizes).  This fails in about 5% of primer sets.
*Generally, 2 distinct PCR products can be amplified in a single reaction.  I do this to check the 5' and 3' ends of an integration in a single reaction (4 primers and different expected product sizes).  This fails in about 5% of primer sets.
==Contact==
==Contact==
[[Special:Emailuser/Tetm2002|Tet]] ([http://biochemistry.ucsf.edu/~blackburn/ Blackburn Lab])
[[Special:Emailuser/Tetm2002|Tet]] ([http://biochemistry.ucsf.edu/~blackburn/ Blackburn Lab])

Revision as of 21:28, 20 March 2009

Back to Yeast Colony PCR

Back to Protocols

Contents

Overview

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

Materials

Procedure

Yeast Cell Lysis

  1. Aliquot 10uL of 0.02M NaOH into PCR tubes.
  2. Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
    • If the solution is cloudy, you've added enough cells.
    • I have been told adding too much yeast can inhibit the reaction.
  3. 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

  1. 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
  2. Aliquot 9uL of the master mix solution into fresh PCR tubes.
  3. Transfer 1uL of boiled samples to the master mix aliquots (a multi-channel pipette is helpful here).
  4. Run the following PCR cycle:
    1. 5 min at 95C
    2. 30 cycles of:
      1. 10 sec at 95C
      2. 10 sec at 50C (or appropriate annealing temperature)
      3. 1 min/kbp at 72C (I generally do 30 sec)
    3. 10 min at 72C (I don't think this step is critical)

Notes

  • Q-solution is critical for this protocol; the main ingredient is betaine.
  • Note that the primer concentration we use is about ten-times more than standard PCR protocols.
  • The amount of taq used in this updated protocol is more per volume than the original protocol, but still less overall.
  • 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 size should be as short as possible. Anything less than 1kbp can be easily amplified.
  • Generally, 2 distinct PCR products can be amplified in a single reaction. I do this to check the 5' and 3' ends of an integration in a single reaction (4 primers and different expected product sizes). This fails in about 5% of primer sets.

Contact

Tet (Blackburn Lab)

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