E.COLI TRANSFORMATION

1. Thaw plasmid DNA and competent cells on ice.

2. In a 1.5 ml epindorf, alliquot 2 ul of plasmid DNA into 100 ul of competent E.coli cells.

3. Mix gently by inversion or finger tapping (do not vortex).

4. Place tubes on ice for 20 minutes.

5. Heat shock cells by placing the tubes at 42 degrees C for 90 seconds.

6. Place tubes on ice for 3 minutes.

7. Add 1 ml LB to each tube.

8. Allow cells to recover for 30 -60 minutes at 37 degrees C (agitation is optional).

9. Spin cells to a pellet by high speed spin for about 20 seconds.

10. Aspirate all but ~100ul of media.

11. Resuspend cells in remaining media and plate full volume onto 150mm LB +AMP plates.

12. Score colonies 12-36 hours after plating.