Transformation of Bacteria for DNA or protein Expression (EAH 10/01)

1) Remove cells from the –80 and place directly into your ice bucket. Thaw on ice 15 minutes.

2) Add DNA from plasmid to be transformed. For ligations use 10uL of ligation reaction per 50-100uL of cells. For purified plasmid, use 0.1ug per aliquot of cells. Do not mix or stir the cells they are very fragile.

FOR CHEMICALLY COMPETENT CELLS:

3) Incubate cells on ice with the DNA for 30 minutes, in the meantime make sure there is a 42°C water bath/block that has water in it.

4) Bring ice bucket with cells and a timer over to the water bath/heat block. Immerse tube of cells in 42°C bath for 45 seconds.

5) Remove tube from water bath and place directly on ice to recover for 2 minutes.

6) Add 800uL of sterile LB (no antibiotics) and grow with shaking for 1 hour at 37°C.

7) Spin cells in eppendorf fuge for 1 minute at high speed, remove 700uL of media and resuspend the cell pellet in the remaining media in the tube. Plate this onto the correct antibiotic LB plate.

FOR ELECTROCOMPETENT CELLS:

3) Take an aliquot of cells 40-50uL and incubate with 0.5 uL of 0.1-0.5ug of DNA on ice for 5 minutes. In the meantime add a ethanol sterilized cuvette on ice.

4) Wipe off cuvette sides so that the currrent does not Arc. Place into cuvette holder of electroporator and set voltage to 1.75V and time for 1s.

5) Press dots to start- when you hear a beep then you are done, add 750uL LB to get cells out of cuvette. If it is pure plasmid you can just plate onto LB, for ligations, recover for one hour at 37°C with shaking and then follow step 7 (above). Although I prefer chemically competent cells for ligations.