Protocol F.3

Frozen Competent E. coli Preparation and Transformation

This is a very easy protocol which yields transformation efficiencies appropriate for routine cloning and transformation.

 

Solutions

LB

10 g Tryptone

5 g Yeast Extract

5 g NaCl

1 ml 1N NaOH

up to 1 liter with Q

Autoclave and store at 4°C

100 mM CaCl2

14.7 g CaCl2

up to 1 liter with Q

Autoclave and store at 4°C

4:1 CaCl2:Glycerol

40 ml 100 mM CaCl2

10 ml glycerol

autoclave and store at 4°C

Also Needed:

sterile eppendorf tubes, sterile 250 ml conical bottles, repeat pipeter

 

Procedure

• Inoculate a 25 ml LB overnight culture from a colony-purified fresh streak of a frozen stock of the desired strain.

• Inoculate 500 ml LB in a 2 liter flask with the 25 ml saturated culture and monitor the OD600 in 1 hour and every half hour thereafter until it reaches 0.6 to 0.7. Harvest the cells by spinning in a J6B centrifuge in 2 250 ml conical bottles (Corning) at 3,000 rpm for 10 minutes at 4°C.

• Discard the LB and gently resuspend each bacterial pellet in 25 ml ice cold 100 mM CaCl2. Spin again at 3,000 rpm for 10 minutes at 4°C.

• Discard the supernatant and gently resuspend each bacterial pellet in 25 ml ice cold 100 mM CaCl2. Hold overnight on ice.

• Spin at 3,000 rpm for 5-10 minutes at 4°C and gently resuspend each pellet in 5 ml 4:1 CaCl2:Glycerol.

• Aliquot using the repeat pipeter and freeze in liquid nitrogen. Store at -80°C. I usually get 1x106-1x107 cfu/mg of plasmid DNA.

• To transform mix 50 ml of frozen competent cells with the DNA and hold on ice for 10 minutes.

• Incubate at 37°C for 5 minutes or 42°C for 1 minute and hold on ice for another 10 minutes.

• Inoculate 1 ml LB or SOC and shake at 37°C for 20-30 minutes.

• Streak/spread 100 ml.

• Alternately, add the DNA directly to the frozen competent E. coli and incubate at 37° for 5 minutes. Plate immediately. This transformation procedure works just as well as the longer procedure described above.