Yu:Lysis

From OpenWetWare

Jump to: navigation, search

Home       Procedures       | Lab Members       | Publications       | Research Interests       | Internal Database       Image:UBLogo.jpg


Quick Yeast Lysis Procedure
1. Grow yeast cells to late-log phase Grow yeast
2. Pellet 2mL cell culture in fast-prep tube. Remove excess supernatant. 2mL yeast culture pelleted in fastprep tube
3. Prepare 600μL completed yeast lysis buffer per fastprep tube. Multiply each ingredient by the number of fastprep tubes to be lysed. Keep on ice after prepared and while using. Prep completed lysis buffer per tube:
Completed lysis buffer
PBSMT
5μL 200mM PMSF per 1mL PBSMT
2.6μL .5M Benzamide per 1mL PBSMT
1μL PLAAC per 1mL PBSMT
4. Add 100μL complete lysis buffer to each fastprep tube. Keep on ice or in cold room for remaining steps. 100μL comp. lysis buffer per tube
5. Add approximately 100μL acid-washed glass fastprep beads to each tube. 100μL beads per tube
6. Fastprep at 6.5 m/s for 30 seconds Fastprep at 6.5 for 30s
7. Add 500μL completed lysis buffer to each fastprep tube 500μL comp. lysis buffer per tube
8. Spin at top speed for 10 minutes at 4°C Spin top speed 10 minutes at 4°C
9. Pipette off supernatant, which contains lysates. Store on ice or in -20°C freezer. Remove and save supernatant
Personal tools