1) Inoculate 100 mL of autoclaved LB medium (add antibiotic as appropriate for the host strain) from a freezer stock or from a plate containing a single colony.

2) Monitor the optical scattering at 600 nm. When this reaches approximately 0.3 (go no lower than 0.25; no higher than 0.35), tranfer the culture to two 50 mL sterile falcon tubes.

3) Incubate the cells on ice for 30 minutes.

4) Spin the cells: 4 degrees, 7 krpm, 7 minutues in Beckman FA0850 rotor.

5) Decant the supernatant.

6) Gently resuspend the cells in 2.5 mL ice-chilled sterile 0.1 M CaCl2.

7) Incubate the cells on ice. The tranformation efficiency will increase, reaching a maximum after approximately 12 - 16 hours.


Tranformation of Ligation Mixture

1) Chill the ligation mixture on ice.

2) Transfer 200 uL of the above chemically competent cells for each 10 uL of the ligation mixture.

3) Mix gently. Incubate 30 minutes on ice.

4) Heat shock by transferring to a 45 degrees water bath (typically we use heat block with a small amount of water added to the the wells) for 45 SECONDS!

5) Immediately return the tube to the ice bath. Incubate 2 minutes on ice.

6) Add 800 uL of sterile LB (be certain that this is not contaminated - its best to use a fresh tube!).

7) Incubate at 37 degrees to allow the cells to express their antibiotic resistance. For ampicillin, this is fast, and takes no longer than 30 minutes. For kanamycin, this is slow, and can take up to an hour or more. See the cloning manual (Maniatis) for further details.

8) Next, gently pellet the cells (7 krpm, 1 minute in eppendorf tabletop centrifuge) and remove the supernatant.

9) Gently resuspend the cells in LB (again, be certain to use LB that is not contaminated - its best to use a fresh tube if you are not sure).

10) Plate 25 uL and 100 uL of the cells onto a LB plate containing the appropriate antibiotic to be used for the selection.