IIT Madras

And then the E.coli said, "Let there be light"

Preperation of competent cells - DH5alpha

• Autoclave 0.1 M CaCl2 in 20% glycerol.
• Primary culture in 5 ml of LB broth for 16 hours.
• Add 1 ml of primary culture into 50 ml of media for secondary culture.
  Check OD at 600 nm.
• Incubate secondary cultures at 37 C in a shaker.
• Check OD600 after 1 hour, 1.5 hours, 2 hours.
• Keep the cells on ice (for 10 minutes) for harvesting as soon as OD600 reaches 0.4 (0.4 -0.6 is the range).
• Pellet down the cells at 6000 rpm for 10 min (4 C ).
  (Each 50 ml culture can be added to 50 ml falcon tubes)
• Add 10 ml of ice cold 0.1M CaCl2.
• Resuspend the pellet and keep on ice for 10 minutes.
• Centrifuge at 6000 rpm for 10 min (4 C).
• Resuspend the pellet in 5 ml ice cold 0.1 M CaCl2 Solution.
• Again, keep on ice for 10 min. and centrifuge at 6000 rpm for 10 min.
• . Resuspend the pellet in 2 ml ice cold 0.1 M CaCl2 Solution. Alliquot 100 ul into a number of tubes and store them at -80 C.

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