Subculturing E. Coli
What you need:
- Autoclaved 250 mL flasks
- Liquid Media
- Antibiotic, if necessary (not for DH5a c competent cells)
- An overnight culture of the cells of interest
Procedure:
1. Into your autoclaved flask add:
- 50 mL liquid media
- 50 ul antibiotic solution if necessary
- 500 uL overnight liquid culture.
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.
2. Incubate in the 37˚C warm room on a stirrer for 4-6 hours
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.
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