Materials Required:-
- LB broth
- Culture plates
- Ice cold CaCl2.2H2O (1 M)
- Ice cold MgCl2 CaCl2 solution
- Shaking incubator
- Vortex mixer
- Centrifuge
- Water bath
- Inoculation loop
- Microfuge tubes
- Polypropylene tubes
- Micro pipettes and tips
Procedure:-
- Pick a single bacterial colony from a culture plate which is incubated for 16-20 hours at 37°C. Transfer the colony into 100 ml LB broth a 1-liter flask. Incubate the culture for 3 hours at 37°C with vigorous agitation, monitoring the growth of the culture. As a guideline, 1 OD600 of a culture of E. coli strain DH 5 alpha contains approx. 109 bacteria/ml.
- Transfer the bacterial cells to sterile, disposable, ice-cold 50ml polypropylene tubes. Cool the cultures to 0°C by storing them on ice for 10 minutes.
- Recover the cells by centrifugation at 2700g at 4°C for 10 minutes .
- Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.
- Resuspend each pellet by swirling or gentle vortexing in 30 ml of ice-cold MgCl2-CaCl2 solution.
- Recover the cells by centrifugation at 2700g at 4°C for 10 minutes .
- Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.
- Resuspend the pellet in 2 ml of ice-cold 0.1 M CaCl2 (or TFB) by gentle vortexing for each 50ml of original culture. Standard TFB may be used instead of CaCl2 for most strains of E. coli.
- At this point, either use the cells directly for transformation or dispense into aliquots and freeze at -70°C.
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