UA Biophysics:Protocols:Competent Cells
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COMPETENT CELLS PROTOCOL
- 1.Grow an ON in LB media (the day before)
- 2.Dilute the ON at least 1/100, into 20-25mL in an 200mL Erlenmeyer
- 3.Grow the diluted bacteria until they reach an OD600 between 0.2 and 0.5
- 4.Put the eppendorfs in ice, to make sure they are cold
- 5.Check that the TSS Buffer is also cold
- 6.Take the culture and separate it into two 50mL falcon tubes.
- 7.Leave the falcon tubes on ice during 10 min.
**The next steps should be carried out at 4°C and cells should be left on ice whenever possible**
- 8.Centrifuge during 10 min at 3000rmp and 4°C
- 9.Remove the supernatant and make the best effort to remove all the remaining media by using pipettes.
- 10.Resuspend the centrifuged cells in TSS Buffer. The recommended amount of TSS Buffer for this step is about 10% of the volume that was centrifuged.
- 11.Distribute the resuspended volume into eppendorfs in aliquots of 100uL.
- 12.Store the competent cells in a -80°C freezer.
- Sofia Alfonso-Sanchez 00:28, 1 August 2015 (EDT):