Smolke:Protocols/Yeast Colony PCR
Colony PCR from yeast, either to check inserts etc. or for sequencing.
Travis' Version (Lyticase lysis)
The basic idea is breaking the cells with lyticase and heat, then doing PCR.
Josh's Version (NaOH lysis)
Genomic Prep by Harju Bust'n'Grab
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
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.