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Team:UIUC-Illinois/Notebook/Protocols/Subculturing
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| - | <center><h1> | + | <center><h1>Subculturing E. Coli</h1></center> |
| + | <br/> | ||
| + | What you need: <br/><br/> | ||
| + | - Autoclaved 250 mL flasks <br/> | ||
| + | - Liquid Media <br/> | ||
| + | - Antibiotic, if necessary (not for DH5a c competent cells) <br/> | ||
| + | - An overnight culture of the cells of interest <br/><br/> | ||
| + | |||
| + | Procedure: <br/><br/> | ||
| + | |||
| + | 1. Into your autoclaved flask add:<br/><br/> | ||
| + | - 50 mL liquid media <br/> | ||
| + | - 50 ul antibiotic solution if necessary <br/> | ||
| + | - 500 uL overnight liquid culture. <br/><br/> | ||
| + | |||
| + | Note: there is a 1:100 ratio of cells to media. Furthermore there is a 1:1000 ratio of antibiotic to media. When subculturing DH5alpha, antibiotic solutions are not required. <br/><br/> | ||
| + | |||
| + | 2. Incubate in the 37˚C warm room on a stirrer for 4-6 hours <br/><br/> | ||
| + | Note: Need to check the cells 2 hours after inoculation to test their optical density (OD). The best competent cells come from cells in exponential phase of their growth. Therefore an OD of 0.4-0.8 is desired and this happens after 2-4 hours of incubation. <br/><br/> | ||
| + | |||
</div> | </div> | ||
Latest revision as of 15:31, 4 June 2012
