Smolke:Protocols/Yeast Colony PCR

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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 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 -80oC, 5 min • Thaw at 95oC, 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 -80oc, 5 min • Spin 5 min at 4oC • Wash pellet with 70% ethanol • Re-suspend in 50 µl TE - use 1 µl in a PCR reaction 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 Beads Materials Procedure Adapted by Mike from protocol from friend in the Herschlag group: 1. Grow 5-10 ml culture of yeast to saturation. 2. Collect cells by centrifugation in 15 ml conical tube (~3000xg, 5-10 min). 3. Aspirate supernatant and resuspend in 500 µl of ddH2O. 4. Transfer cells to 1.5 ml eppendorf tube. 5. Centrifuge at 3500rcf at room temp for 3.5 min. 6. Decant. 7. Pipette briefly to resuspend pellet in residual liquid. 8. Add 200 ul detergent lysis buffer, ~100 mg (50uL) glass beads. 9. Tape to vortex machine in cold room, vortex for 10-20min. 10. Add 200 ul PCI (phenol:chloroform:isoamyl alcohol:TE; 25:24:1), vortex briefly. 11. Centrifuge at max for 5 min (4C). 12. Transfer aqueous top layer to fresh eppendorf tube. 13. Add 1 ml ice cold 100% EtOH + 50 ul 3M sodium acetate. 14. Spin for 10-20min high speed @ 4C. 15. Decant off liquid and vacufuge until pellet dries. 16. Resuspend in water, 1x TE buffer pH 8.0, or EB buffer ~100ul 17. Test variety of dilutions for PCR (1 ul of 1:10 or 1:100 as template for PCR often give good results) – unfortunately, this is somewhat empirical based upon the strain, shearing, and how well your genomic isolation went. Note: Detergent lysis buffer is 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA.