Smolke:Protocols/Yeast Colony PCR
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: 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. |