Chemically competent cells preparation

Reagents:

1. CaCl2 80mM/Tris HCl 10mM.

2. CaCl2 100mM/glicerol 15%.

3. Luria-Bertani Medium (LB).

Steps:

Pre-inoculum

1. Inoculate bacteria in 50 ml of liquid LB medium.

2. Leave at 37 °C in agitation overnight.

Inoculum

1. Add 1 ml of pre-inoculum into 50ml of liquid LB medium.

2. Leave at 37 °C in shaker for 1 hour to 2 hours, depending on cell growth.

3. Achieving absorbance at 600nm spectrophotometer of 0.2 - 0,4abs.

4. Leave on ice for 30 minutes.

Cell preparation

All materials used in this step must be properly chilled.

1. Add 50ml of culture in Tubes (50ml) and centrifuged for 10 minutes at 4 °C in 4.000 rpm.

2. Discard supernatant and resuspend in 1ml of CaCl2 80mM/Tris HCl. After that, completing the volume to 20ml. Incubate on ice for 15 minutes.

3. Centrifuge for 10minutes at 4 °C in 4,000 rpm.

4. Discard supernatant.

5. Resuspend the pellet with 1ml CaCl2 100mM/15% glycerol.

6. Aliquot 50ul of cell.