Smolke:Protocols/Yeast Colony PCR

Home        Contact        Internal        Protocols        Lab Members        Publications        Research       

Overview Colony PCR from yeast, either to check inserts etc. or for sequencing. Travis' Version (Lyticase lysis) 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. Dilute stock of lyticase to 50 U/mL in TE. 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 Incubate at 37°C for 30 min Incubate at 95°C for 10 min 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 (NaOH lysis) 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. Genomic Prep by Harju Bust'n'Grab Materials overnight yeast cultures lysis buffer - 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0) phenol:chloroform chloroform ethanol (70% and 100%) sodium acetate TE Procedure Adapted by Kate from Harju et al., 2004 Spin down 1.5 ml O/N culture and remove media Re-suspend in 200 µl lysis buffer and transfer to a thin-walled PCR tube Freeze at -80 °C, 5 min Thaw at 95 °C, 1 min on PCR block Repeat once and transfer to 1.5 mL eppendorf tube Vortex vigorously Add 200 µl phenol:chloroform and vortex well Spin 2 min, remove aqueous phase Add 200 µl chloroform and vortex well Spin 2 min, remove aqueous phase Add 400 µl ice-cold ethanol and 40 µl sodium acetate Precipitate at -80 °C, 5 min Spin 5 min at 4 °C Wash pellet with 70% ethanol Re-suspend in 50 µl TE - use 1 µl in a PCR reaction Genomic Prep with Glass Beads 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 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; 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. Notes Yeast colony PCR works best on freshly grown colonies, immediately after transformation.