Preparation of fresh competent E. coli for transformation using CaCl2
1. Seed culture :
(1) Pick a single colony from a plate fresh grown for 16-20 h at 37 C and transfer it
into 3 ml of LB broth in sterile 15-ml polypropylene tube.
(2) Incubate the culture overnight at 37 C with vigorous shaking (>250 cycles/min in
a rotary shaker).
2. Main culture :
(1) Inoculate 500 l of seed culture into 50 ml of LB broth in sterile 250-ml flask.
(2) Incubate the culture for about 2 h at 37 C with vigorous shaking (>250 cycles/min
in a rotary shaker) until the OD600 reaches 0.3-0.4 (< 108 cells/ml).
 Aseptically transfer the cells to sterile, ice-cold 50-ml polypropylene tube and cool
the cultures to 0 C by storing the tube on ice for 10 min.
 Centrifugation at 4,000 rpm for 15 min at 4 C.
 Decant the media from the cell pellets and stand the tube in an inverted position for 1
min to allow the last traces of media to drain away.
 Resuspend the cell pellets in 10 ml of filter-sterilized ice-cold 0.1 M CaCl2 and store
for 5 min on ice. 
 Centrifugation at 4,000 rpm for 15 min at 4 C.
 Decant the media from the cell pellets and stand the tube in an inverted position for 1
min to allow the last traces of media to drain away.
 Resuspend the cell pellets in 2 ml of filter-sterilized ice-cold 0.1 M CaCl2 and store
for 2-3 h on ice.
 Transfer 200 l of suspension of competent cells to a chilled sterile microcentrifuge
cells and continue to transformation or freeze immediately and store at –70 C.
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Dr. Lee’s Lab