Yu:Lysis: Difference between revisions
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quick lyse |
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|2mL yeast culture pelleted in fastprep tube | |2mL yeast culture pelleted in fastprep tube | ||
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|3. Prepare | |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. | ||
|valign="top"|Prep completed lysis buffer per tube: | |||
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Multiply each ingredient by the number of fastprep tubes to be lysed. Keep on ice after prepared and while using. | |+ '''Completed <span id="buffer">lysis buffer</span>''' | ||
|Prep completed lysis buffer per tube: | |align="left"|[[Yu:PBS#PBSMT|PBSMT]] | ||
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|5μL 200mM [[PMSF]] per 1mL PBSMT | |||
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|2.6μL .5M Benzamide per 1mL PBSMT | |||
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|1μL [[Yu:PLAAC|PLAAC]] per 1mL PBSMT | |||
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|4. Add 100μL complete lysis buffer to each fastprep tube. Keep on ice or in cold room for remaining steps. | |4. Add 100μL complete lysis buffer to each fastprep tube. Keep on ice or in cold room for remaining steps. | ||
Latest revision as of 00:21, 18 December 2009
| 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: | ||||
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| 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 | ||||
