Yu:Lysis: Difference between revisions
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(quick lyse) |
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|3. Prepare 800μL completed yeast lysis buffer per fastprep tube: | |3. Prepare 800μL completed yeast lysis buffer per fastprep tube: | ||
* | *600μL 1x[[Yu:PBS#PBSMT|PBSMT]] | ||
* | *3μL PMSF | ||
* | *1.56μL .5M Benzamide | ||
*0. | *0.6μL PLAAC | ||
Multiply each ingredient by the number of fastprep tubes to be lysed. Keep on ice after prepared and while using. | 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: | |valign="top"|Prep completed lysis buffer per tube: | ||
* | *600μL 1x[[Yu:PBS#PBSMT|PBSMT]] | ||
* | *3μL PMSF | ||
* | *1.56μL .5M Benzamide | ||
*0. | *0.6μL PLAAC | ||
|- | |- | ||
|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. | ||
Revision as of 15:43, 10 June 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 800μL 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:
|
| 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 |
