Blackburn:Yeast Colony PCR

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==Notes==
==Notes==
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*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])
[[Special:Emailuser/Tetm2002|Tet]] ([http://biochemistry.ucsf.edu/~blackburn/ Blackburn Lab])

Revision as of 00:28, 12 June 2006

Contents

Overview

This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.

Materials

Procedure

Yeast Cell Lysis

  1. Aliquot 3uL 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.
  3. [optional] Quick-freeze the cells in liquid nitrogen
    • I normally skip this step. Works just fine.
  4. 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.

PCR

  1. 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
  2. Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume).
  3. Run the following PCR cycle:
    1. 5 min at 94C
    2. 30 cycles of:
      1. 30 sec at 94C
      2. 30 sec at 55C (or appropriate annealing temperature)
      3. 1 min/kbp at 72C
    3. 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

Tet (Blackburn Lab)

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