Week 1

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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.