Team:Trieste/protocols
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<h2 class="notebook_title">Preparation of Competent Cells</h2> | <h2 class="notebook_title">Preparation of Competent Cells</h2> | ||
Work as sterile as possible. | Work as sterile as possible. | ||
- | 1. Take an 100mL aliquot of frozen cells (use DH5-α cells in this case) from the -80°C and inoculated. | + | 1. Take an 100mL aliquot of frozen cells (use DH5-α cells in this case) from the -80°C and inoculated.</br></br> |
- | 2. Grow the cells on a shaker at 37°C until they reach an O.D.600nm=0,6. | + | 2. Grow the cells on a shaker at 37°C until they reach an O.D.600nm=0,6.</br></br> |
- | 3. Transfer in sterile Falcon (50mL). | + | 3. Transfer in sterile Falcon (50mL).</br></br> |
- | 4. Centrifuge it at 4500 rpm for 10 minutes at 4°C. | + | 4. Centrifuge it at 4500 rpm for 10 minutes at 4°C. </br></br> |
- | 5. Resuspend the bacteria pellet on ice in 0,1M of ice cold CaCl2. | + | 5. Resuspend the bacteria pellet on ice in 0,1M of ice cold CaCl2.</br></br> |
- | 6. Keep this suspension on ice for overnight. | + | 6. Keep this suspension on ice for overnight.</br></br> |
- | 7. Centrifuge it at 4500 rpm for 10 minutes at 4°C. | + | 7. Centrifuge it at 4500 rpm for 10 minutes at 4°C.</br></br> |
- | 8. Resuspend the pellet in 8mL of RF2 (MOPS 1mM, RbCl 10mM, CaCl2 75mM, glycerol 15%w/v. | + | 8. Resuspend the pellet in 8mL of RF2 (MOPS 1mM, RbCl 10mM, CaCl2 75mM, glycerol 15%w/v.</br></br> |
- | 9. Dispense in aliquots and freeze cells at -80°C. | + | 9. Dispense in aliquots and freeze cells at -80°C.</br></br> |
</div> | </div> |
Revision as of 16:53, 25 September 2012