Team:ICT-Mumbai/Protocols
Materials
1. Overnight E. coli culture
2. 10 ml LB medium in culture tubes
3. 0.1 M CaCl2
4. 0.1 M CaCl2 containing 25% glycerol
Equipment
1. Spectrophotometer or plate reader
2. Centrifuge
Procedure
1. Subculture 100 µl overnight grown culture of E. coli in 10 ml LB medium. Shake at 200 rpm at 37°C.
2. After ~2 hours, using either a spectrophotometer or a plate reader, measure absorbance of the culture at 600 nm. At OD600 = 0.2, harvest the cells by centrifugation.
3. Wash cells with ice cold solution of 0.1 M CaCl2 two to three times.
4. Resuspend cells in 100 μl of 0.1 M CaCl2 containing 25% glycerol and store at –80°C till further use.
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