Smolke:Protocols/Yeast Colony PCR: Difference between revisions
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==Genomic Prep by Phenol:Chloroform with Glass Beads== | ==Genomic Prep by Phenol:Chloroform with Glass Beads== | ||
===Materials=== | ===Materials=== | ||
*overnight yeast cultures | |||
*lysis buffer - 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA | |||
*phenol:chloroform:isoamyl alcohol | |||
*ice cold 100% EtOH | |||
*glass beads | |||
===Procedure=== | ===Procedure=== | ||
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#Add 200 ul detergent lysis buffer, ~100 mg (50uL) glass beads. | #Add 200 ul detergent lysis buffer, ~100 mg (50uL) glass beads. | ||
#Tape to vortex machine in cold room, vortex for 10-20 min. (You can vortex for 2 min at room temp if you don't need much DNA.) | #Tape to vortex machine in cold room, vortex for 10-20 min. (You can vortex for 2 min at room temp if you don't need much DNA.) | ||
#Add 200 ul PCI (phenol:chloroform:isoamyl alcohol | #Add 200 ul PCI (phenol:chloroform:isoamyl alcohol; 25:24:1), vortex briefly. | ||
#Centrifuge at max for 5 min (4 °C). | #Centrifuge at max for 5 min (4 °C). | ||
#Transfer aqueous top layer to fresh eppendorf tube. | #Transfer aqueous top layer to fresh eppendorf tube. | ||
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#Resuspend in ~100 uL water, 1x TE buffer pH 8.0, or EB buffer. | #Resuspend in ~100 uL water, 1x TE buffer pH 8.0, or EB buffer. | ||
#For PCR use 1 ul of 1:10 or 1:100 dilution of the resuspension as template - this varies depending on the strain, shearing, and how well your genomic isolation went. You can nanodrop the resuspension to estimate the concentration, and use 10-30 ng in a 25 uL PCR reaction. | #For PCR use 1 ul of 1:10 or 1:100 dilution of the resuspension as template - this varies depending on the strain, shearing, and how well your genomic isolation went. You can nanodrop the resuspension to estimate the concentration, and use 10-30 ng in a 25 uL PCR reaction. | ||
Revision as of 21:59, 26 October 2011
OverviewColony PCR from yeast, either to check inserts etc. or for sequencing. Travis' Version (Lyticase lysis)Materials
ProcedureThe basic idea is breaking the cells with lyticase and heat, then doing PCR.
Notes
Josh's Version (NaOH lysis)Materials
Procedure
Genomic Prep by Harju Bust'n'GrabMaterials
ProcedureAdapted by Kate from Harju et al., 2004
Genomic Prep by Phenol:Chloroform with Glass BeadsMaterials
ProcedureAdapted by Mike from protocol from friend in the Herschlag group, and further adapted by Stephanie
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