Transformation

Protocol

1.  Aliquot 33 ul of competent cells (i.e., E. coli DH5alpha) on ice

2.  Add 3.5 ul of DNA (i.e., the ligation products) on ice

3.  Vortex to mix, then immediately on ice

4.  Incubate for 3 - 5 min on ice

5.  Heat shock at 42°C for 60 sec on a dry bath

6.  Recover for 3 - 5 min on ice

7.  Add 500 ul of LB "only" and culture by shaking at 37°C for 60 min in an incubator.

8.  Warm agar plates in the incubator.

9. Centrifuge the bacteria at 15,000 xg and discard *430 ul of the supernatant.

10. Vortex the remaining containing the pellet.

11. Spread onto the LB+Cm (20 ug/ml) agar plate with glass beads.

12. Culture at 37°C O/N (overnight, 16-18 hr) in an incubator.

13. Count and observer the colonies.