UA Biophysics:Protocols:Competent Cells
COMPETENT CELLS PROTOCOL
- Grow an ON in LB media (the day before)
- Dilute the ON at least 1/100, into 20-25mL in an 200mL Erlenmeyer
- Grow the diluted bacteria until they reach an OD600 between 0.2 and 0.5
- Put the eppendorfs in ice, to make sure they are cold
- Check that the TSS Buffer is also cold
- Take the culture and separate it into two 50mL falcon tubes.
- Leave the falcon tubes on ice during 10 min.
- The following steps should be carried out at 4°C and cells should be left on ice whenever possible
- Centrifuge during 10 min at 3000rmp and 4°C
- Remove the supernatant and make the best effort to remove all the remaining media by using pipettes.
- 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.
- Distribute the resuspended volume into eppendorfs in aliquots of 100uL.
- Store the competent cells in a -80°C freezer.