Yu:Lysis
From OpenWetWare
(Difference between revisions)
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Current revision (20:21, 17 December 2009) (view source) m |
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|colspan="2" align="center"| | |colspan="2" align="center"| | ||
{| border="0" cellpadding="5" style="width:50%" | {| border="0" cellpadding="5" style="width:50%" | ||
| - | |+ '''Completed <span id="buffer">lysis buffer</span> | + | |+ '''Completed <span id="buffer">lysis buffer</span>''' |
| - | |align="left"| | + | |align="left"|[[Yu:PBS#PBSMT|PBSMT]] |
|- | |- | ||
| - | |5μL | + | |5μL 200mM [[PMSF]] per 1mL PBSMT |
|- | |- | ||
| - | |2.6μL | + | |2.6μL .5M Benzamide per 1mL PBSMT |
|- | |- | ||
| - | |1μL | + | |1μL [[Yu:PLAAC|PLAAC]] per 1mL PBSMT |
|} | |} | ||
|- | |- | ||
Current revision
| 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 | ||||
