PREPARATION OF COMPETENT CELLS (Sambrook et al.

2001)

1. Frozen bacterial stock is thawed at 37°C and streaked on LB agar plates using sterile

platinum loop.

2. Incubate at 37°C for 16 to 18 hours.

3. Single colony from the plate is inoculated into 25 ml LB broth.

4. Incubate at 37°C for 16 to 18 hours.

5. At the end of incubation, 2.5 ml of this culture is transferred to a 250 ml LB broth.

6. Incubate at 37°C in shaking incubator ubtil the culture attains OD of 0.3 to 0.4 at A600 (3-4

hrs incubation).

7. The culture is chilled on ice.

8. Pellet cells by centrifugation at 6000 X g at 4°C for 5 min.

9. Resuspended the cell pellet in 25 ml of ice cold 100 mM CaCl2.

10. Incubate on ice for 30 min.

11. Pellet the cells by centrifugation 6000 X g for 5 min at 4°C.

12. Resuspend the cells (pellet) in 1.75 ml of ice cold 100 mM CaCl2 + 0.75 ml of sterile 50%

glycerol.

13. The cell suspension is thoroughly but gently mixed and aliquoted (150 µl) in 1.5 ml micro

centrifuge tubes and stored at -70°C.