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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. | ||
| + | 1. Take an 100mL aliquot of frozen cells (use DH5-α cells in this case) from the -80°C and inoculated. | ||
| + | |||
| + | 2. Grow the cells on a shaker at 37°C until they reach an O.D.600nm=0,6. | ||
| + | |||
| + | 3. Transfer in sterile Falcon (50mL). | ||
| + | |||
| + | 4. Centrifuge it at 4500 rpm for 10 minutes at 4°C. | ||
| + | |||
| + | 5. Resuspend the bacteria pellet on ice in 0,1M of ice cold CaCl2. | ||
| + | |||
| + | 6. Keep this suspension on ice for overnight. | ||
| + | |||
| + | 7. Centrifuge it at 4500 rpm for 10 minutes at 4°C. | ||
| + | |||
| + | 8. Resuspend the pellet in 8mL of RF2 (MOPS 1mM, RbCl 10mM, CaCl2 75mM, glycerol 15%w/v. | ||
| + | |||
| + | 9. Dispense in aliquots and freeze cells at -80°C. | ||
| + | |||
</div> | </div> | ||
Revision as of 16:51, 25 September 2012
The Jolly JoCare
a safe probiotic platform for protein expression
Week 1
More
Preparation of Competent Cells
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. 2. Grow the cells on a shaker at 37°C until they reach an O.D.600nm=0,6. 3. Transfer in sterile Falcon (50mL). 4. Centrifuge it at 4500 rpm for 10 minutes at 4°C. 5. Resuspend the bacteria pellet on ice in 0,1M of ice cold CaCl2. 6. Keep this suspension on ice for overnight. 7. Centrifuge it at 4500 rpm for 10 minutes at 4°C. 8. Resuspend the pellet in 8mL of RF2 (MOPS 1mM, RbCl 10mM, CaCl2 75mM, glycerol 15%w/v. 9. Dispense in aliquots and freeze cells at -80°C.Transformation - Heat Shock
Miniprep
Gel Extraction
Clean colony PCR
E.L.I.S.A.
Western blot
Stripping
