Difference between revisions of "Smolke:Protocols/Yeast Colony PCR"

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==Overview==
 
==Overview==
 
 
Colony PCR from yeast, either to check inserts etc. or for sequencing.
 
Colony PCR from yeast, either to check inserts etc. or for sequencing.
  
==Materials==
+
==Travis' Version==
 
+
===Materials===
 
*Lyticase (from Sigma)
 
*Lyticase (from Sigma)
 
*TE
 
*TE
 
*PCR buffers, primers, polymerase, etc.
 
*PCR buffers, primers, polymerase, etc.
  
==Procedure==
+
===Procedure===
 
The basic idea is breaking the cells with lyticase and heat, then doing PCR.
 
The basic idea is breaking the cells with lyticase and heat, then doing PCR.
 
  
 
#Dilute stock of lyticase to 50 U/mL in TE.
 
#Dilute stock of lyticase to 50 U/mL in TE.
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#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
  
 
+
===Notes===
==Notes==
 
 
*I use this protocol to PCR off the chromosome for sequencing... I clean up the rxn with a Zymoclean kit afterwards.
 
*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.
 
*The amount of yeast doesn't seem to matter much.  I get success with very small colonies or with loads of cells.
  
==Contact==
+
==Josh's Version==
[[User:Tsbayer|Travis]]
+
===Materials===
 +
*20 mM NaOH
 +
*PCR materials
 +
 
 +
===Procedure===
 +
#Aliquot 20uL NaOH into PCR tubes
 +
#Pick colonies (I use pipet tips) into the NaOH
 +
#Incubate at 95C for ~45 minutes
 +
#Centrifuge at max speed for 10 minutes (helps to use plastic inserts in the microfuge)
 +
#Use 1uL of supernatant as template in a (10uL) PCR.

Revision as of 14:12, 20 October 2009

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Overview

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

Travis' Version

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.

Josh's Version

Materials

  • 20 mM NaOH
  • PCR materials

Procedure

  1. Aliquot 20uL NaOH into PCR tubes
  2. Pick colonies (I use pipet tips) into the NaOH
  3. Incubate at 95C for ~45 minutes
  4. Centrifuge at max speed for 10 minutes (helps to use plastic inserts in the microfuge)
  5. Use 1uL of supernatant as template in a (10uL) PCR.