Team:IIT Madras/Notebook/Protocols/Preperation of competent cells
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<h3 style="color:white;">And then the E.coli said, "Let there be light"</h3></div> | <h3 style="color:white;">And then the E.coli said, "Let there be light"</h3></div> | ||
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- | < | + | <h1>Preperation of competent cells - DH5alpha</h1><br/><br/> |
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<li>Autoclave 0.1 M CaCl2 in 20% glycerol.</li> | <li>Autoclave 0.1 M CaCl2 in 20% glycerol.</li> |
Latest revision as of 12:54, 2 October 2011
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.