Smolke:Protocols/Yeast Colony PCR

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#Aliquot lyticase in 50uL quantities
#Aliquot lyticase in 50uL quantities
#Pick colonies (I use a pipette tip) and add to lyticase aliquots, pipette up and down or agitate to break up colony
#Pick colonies (I use a pipette tip) and add to lyticase aliquots, pipette up and down or agitate to break up colony
-
#Incubate at 37* for 30 min
+
#Incubate at 37°C for 30 min
-
#Incubate at 95* for 10 min
+
#Incubate at 95°C for 10 min
#Use as template for PCR - I use 5uL of the cells in a 50uL PCR reaction
#Use as template for PCR - I use 5uL of the cells in a 50uL PCR reaction

Revision as of 15:24, 8 September 2006

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Contents

Overview

Colony PCR from yeast, either to check inserts etc. or for sequencing.

Materials

  • Lyticase (from Sigma)
  • TE
  • PCR buffers, primers, polymerase, etc.

Procedure

The basic idea is breaking the cells with lyticase and heat, then doing PCR.


  1. Dilute stock of lyticase to 50 U/mL in TE.
  2. Aliquot lyticase in 50uL quantities
  3. Pick colonies (I use a pipette tip) and add to lyticase aliquots, pipette up and down or agitate to break up colony
  4. Incubate at 37°C for 30 min
  5. Incubate at 95°C for 10 min
  6. Use as template for PCR - I use 5uL of the cells in a 50uL PCR reaction


Notes

  • I use this protocol to PCR off the chromosome for sequencing... I clean up the rxn with a Zymoclean kit afterwards.
  • The amount of yeast doesn't seem to matter much. I get success with very small colonies or with loads of cells.

Contact

Travis

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