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:-

1. 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.
    
2. 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.
    
3. Recover the cells by centrifugation at 2700g  at 4°C for 10 minutes .
    
4. 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.
    
5. Resuspend each pellet by swirling or gentle vortexing in 30 ml of ice-cold MgCl₂-CaCl₂ solution.
    
6. Recover the cells by centrifugation at 2700g  at 4°C for 10 minutes .
    
7. 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.
    
8. 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.
    
9. At this point, either use the cells directly for transformation or dispense into aliquots and freeze at -70°C.

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