Preparation of Competent Cells

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

Transformation - Heat Shock

1. 1. Take competent E.coli cells from –80°C freezer and place on ice. Allow cells to thaw.
2. 2. Mix cells by flicking the tube gently, then remove 100μl per transformation into a sterile pre-chilled (on ice) 1,5 tube.
3. 3. Add 7μl of DNA per 100μl cells. Quickly flick the tube several times to ensure the even distribution of DNA.
4. 4. Immediately place tubes on ice for at least 30 minutes.
5. 5. Heat shock the cells for 90 seconds in a water bath at exactly 42 °C. Do no shake.
6. 6. Immediately place tubes on ice for 2 minutes. 7. Add 1mL of room temperature LB media (with no antibiotic added) and incubate for 1 hour in shaker at 37°C.Can incubate tubes for 30 minutes with appropriate antibiotic added – usually Ampicillin or Kanamycin.
7. 8. Spread about 100μL of the resulting culture on LB plates - Grow overnight (O/N). The cells may be pelleted by centrifugation at 500 x g for 5 minutes, then the cells can be resuspended and plated.
8. 9. Pick colonies about 12-16 hours later.

Gel Extraction

Clean colony PCR

E.L.I.S.A.

Western blot

Stripping