Preparation of Ca-Cl2 heat-shock competent cells
1. Inoculate a single colony of E. coli cells in 5ml LB. Grow overnight at 37C.
2. Inoculate 50ml of LB with 1:100 dilution of overnight culture. Grow at 37C to an
OD590 0.375-.4
3. Place cells on ice for 5-10min. (Cells need to be kept on ice or at 4C for the rest
of the prep).
4. Centrifuge cells 7min at 1600xg, 4C without BRAKE.
5. Pour off supernatant and resuspend in 10ml ice-cold CaCl2
6. Centrifuge 5min at 1100xg, 4C without brake
7. Pour off supernatant and resuspend each pellet in 2ml CaCl2
8. Dispense cells 100-50ul into prechilled eppendorfs, snap freeze to store.
CaCl2 solution
60mM CaCl2
15% glycerol
10mM PIPES [piperazine-N,N’-bis(2-hydroxypropanesulfonic acid)], pH71
Filter sterilize
Transformation
1.
2.
3.
4.
1
Rapid-thaw cells in hand and place on ice for 10min with DNA (10-100ng).
Heat shock cells by placing tubes in 42C water bath for 2min.
Add 1ml LB and place on wheel for 1hr @ 37C.
Plate aliquots or spin down entire tube at 6-8000 rpm, resuspend in 200ul and
plate on appropriate antibiotic containing plates.
NOT NECESSARY
Created by Dwayne Taliaferro…If this doesn’t work for you feel free to try something else.