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 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. 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.
8. 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.
9. 9. Pick colonies about 12-16 hours later.

Clean colony PCR

1. 1. Pick the bacterial colonies and release them in 50μL distillate/autoclaved water.
2. 2. Boil the sample at 95°C for 5 minutes.
3. 3. Prepare 28μL of mix-PCR solution for each sample (6μL Buffer Taq 5x, 1,8μL MgCl2 25mM, 0,6μL dNTPs 5mM, 0,15μL per primers, 0,15μL Taq polymerase, 19,15μL H2O), blend it and then spin it.
4. 4. Take 2mL of the sample and release into mix-PCR solution and blend it.
5. 5. Impost the PCR machine for 30μL volume and for 30 cycles, 5 minutes at 93°C, 30 seconds at 95°C, 30 seconds at 53°C, 1 minute at 72°C, indefinitely at 4°C.
6. 6. Insert the samples and start the PCR machine.
7. 7. At the end of the PCR the samples are ready for electrophoresis.

E.L.I.S.A.

1. 1. Coat 96-well ELISA plate with 100μL per well of antibody I anti6HIS used 1μg/mL for selection. Coating is in 100mM sodium hydrogen carbonate, pH 9.6. Leave O/N at 25°C.
2. 2. Rinse wells 5x with BSA-PBS 0,1%.
3. 3. Add the bacteria transformed that express the 6HIS tag in different concentration: 10⁶, 10⁵, 10⁴ in different wells. Then in different wells too add bacteria non-transformed in different concentration: 10⁶, 10⁵, 10⁴.
4. 4. Rinse wells 5x with LB media.
5. 5. Add 200μL of LB media and possibly antibiotics. Incubate O/N at 37°C.
6. 6. Plate and incubate at 37°C until formation of bacterial colonies.

Western blot

Stripping