Revathireddy (Talk | contribs) |
Revathireddy (Talk | contribs) |
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− | <button class="accordion"> | + | <button class="accordion">Preparation of <i>E. coli</i> competent cells</button> |
<div class="panel"> | <div class="panel"> | ||
− | <p | + | <p> <i>Materials</i> |
+ | 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 | ||
+ | |||
+ | <i>Equipment</i> | ||
+ | 1. Spectrophotometer or plate reader | ||
+ | 2. Centrifuge | ||
+ | |||
+ | <i>Procedure</i> | ||
+ | 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. | ||
+ | </p> | ||
</div> | </div> | ||
Revision as of 15:12, 29 October 2017