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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. | + | Work as sterile as possible.</br> |
| - | 1. Take an 100mL aliquot of frozen cells (use DH5-α cells in this case) from the -80°C and inoculated.</ | + | <ol><li>1. Take an 100mL aliquot of frozen cells (use DH5-α cells in this case) from the -80°C and inoculated.</li> <li>2. Grow the cells on a shaker at 37°C until they reach an O.D.600nm=0,6.</li> |
| + | <li>3. Transfer in sterile Falcon (50mL).</li> | ||
| + | <li>4. Centrifuge it at 4500 rpm for 10 minutes at 4°C. </li> <li>5. Resuspend the bacteria pellet on ice in 0,1M of ice cold CaCl<sub>2</sub>.</li> <li>6. Keep this suspension on ice for overnight.</li> <li>7. Centrifuge it at 4500 rpm for 10 minutes at 4°C.</li> <li>8. Resuspend the pellet in 8mL of RF2 (MOPS 1mM, RbCl 10mM, CaCl2 75mM, glycerol 15%w/v.</li> <li>9. Dispense in aliquots and freeze cells at -80°C.</li> | ||
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Revision as of 17:02, 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
