Notebook – Protocol – 9. Cell competent preparation

1. From LB agar plate containing no antibiotics, colony of either E. coli DH5alpha or BL21(DE3) cell was taken and culture in 2 ml LB medium at room temperature for overnight for pre-culture.

2. 900 ul of pre-culture was added into 9 ml of sterilized LB medium in 50 ml polypropylene tube, inside a cleanbench.

3. Tube was shaken for 2 hours at room temperature. Because we have no UV spectrophotometer, OD (optical density) of the main culture could not be measured. However, based on our chief instructor - Dr. Arief experiment, 2 hours shaking at room temperature using the above condition culture was enough to get OD660 nm of about 0.6.

4. In a cleanbench, aliquot @1.5 ml of the main culture into cold 4 polypropylene tubes with size of 2 ml. The tubes were pre-chilled by putting inside refrigerator.

5. Centrifuge the 4 tubes at 6,000 rpm, 10 minutes at 4 oC by putting the small microcentrifuge inside a refrigerator.

6. Inside a cleanbench, the supernatants were discarded.

7. Then @1.5 ml of cold 100 mM MgCl2 was added to each tube. Pellet was suspended.

8. Centrifuge the tubes at 6,000 rpm, 10 minutes at 4 °C.

9. Then @1.5 ml of cold 100 mM CaCl2 was added to each tube. Pellet was suspended.

10. Tubes were incubated on ice for 30 minutes.

11. Centrifuge the tubes at 6,000 rpm, 10 minutes at 4 °C

12. Inside a cleanbench, the supernatants were discarded.

13. Then add @75 ul of cold 100 mM MgCl2 were added to each tube. Pellet were suspended.

14. Cell competent was ready to be used in the same day.

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