Smolke:Protocols/Yeast Colony PCR: Difference between revisions
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===Procedure=== | ===Procedure=== | ||
Adapted by Mike from protocol from friend in the Herschlag group | Adapted by Mike from protocol from friend in the Herschlag group, and further adapted by Stephanie | ||
#Grow 3-10 ml culture of yeast to saturation. (You can even use this protocol on single colonies if you don't need much DNA.) | |||
#Collect cells by centrifugation in 1.5 ml eppendorf tube (max speed, 15 s). | |||
#Aspirate supernatant and resuspend in 500 µl of ddH2O. Pellet, decant. | |||
#Pipette briefly to resuspend pellet in residual liquid. | |||
#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.) | |||
#Add 200 ul PCI (phenol:chloroform:isoamyl alcohol:TE; 25:24:1), vortex briefly. | |||
#Centrifuge at max for 5 min (4 °C). | |||
#Transfer aqueous top layer to fresh eppendorf tube. | |||
#Add 50 ul 3M sodium acetate, then 1 ml ice cold 100% EtOH. | |||
#Spin for 10-20 min at high speed at 4 °C. | |||
#Decant off liquid and vacufuge until pellet dries. | |||
#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. | |||
Note: Detergent lysis buffer is 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA. | Note: Detergent lysis buffer is 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA. |
Revision as of 21:55, 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'GrabMaterialsProcedureAdapted by Kate from Harju et al., 2004
Note: Lysis buffer contains 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0) Genomic Prep by Phenol:Chloroform with Glass BeadsMaterialsProcedureAdapted by Mike from protocol from friend in the Herschlag group, and further adapted by Stephanie
Note: Detergent lysis buffer is 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA. |