What you need:
- 50mL subculture of E.coli cells
- 50mL conicial tubes
- Cold 10% glycerol solution
- Autoclaved 1.5mL centrifuge tubes
Procedure:
1. Divide liquid cell subculture into 50mL conical tubes and centrifuge for 10 minute at 4000rpm
2. Discard supernatant and add 25mL cold 10% glycerol
3. Stir the mixture by mixing and centrifuge for an additional 10 minutes
4. Repeat steps 2-3 two more times
5. Discard the supernatant and resuspend the cells in 1mL 10% glycerol
6. Transfer to 1.5mL centrifuge tubes and centrifuge for 2 minutes
7. Discard the supernantant and resuspend the cells in 2 volumes (cell volumes) 10% glycerol
8. Store at -80˚C
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