Smolke:Protocols/Yeast Colony PCR: Difference between revisions
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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. | ||
== | ==Josh's Version== | ||
===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
OverviewColony PCR from yeast, either to check inserts etc. or for sequencing. Travis' VersionMaterials
ProcedureThe basic idea is breaking the cells with lyticase and heat, then doing PCR.
Notes
Josh's VersionMaterials
Procedure
|