Reagents:

1. Sorbitol 1M

Steps:

1. Grow bacteria (Escherichia coli) to log phase (OD 0.4-0.6).

2. Aliquot 1mL of culture to a 1.5mL tube. Make as many aliquots as you need for the number of plasmids you need to transform.

3. Centrifuge aliquots at 12,000g for 1 minute.

4. Remove media and wash with 0.33 volumes (330μL) of 1M sorbitol.

5. Centrifuge at 12,000g for 1 minute.

6. Remove wash and resuspend in 0.05 volumes (40μL) 1M sorbitol.

7. Place tubes in ice and use within 15 minutes.

8. Add plasmid DNA to the tube.

9. Incubate on ice for 30 minutes.

10. Electroporate at 1.900V.

11. Flush cuvette gently with 500ul of LB broth.

12. Allow to recover for 1-2 hrs at 37°C, shake at 200rpm.

13. Spin down cells briefly, pour off most of supernatant, gently resuspend cells and plate.

14. Incubate the plate at 37°C for 12-14 hours.

Freezing these cells for later use is not recommended. Even with storage at -80°C, subsequent transformations often fail.