UA Biophysics:Protocols:Competent Cells: Difference between revisions
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== | ==Material== | ||
* [https://openwetware.org/wiki/TSS TSS Buffer ] | |||
* Overnight (E.coli MG1655) | |||
* LB 25mL in a 200mL Erlenmeyer | |||
* Sterile epperdorfs | |||
==Protocol== | |||
#Grow an ON in LB media (the day before) | #Grow an ON in LB media (the day before) | ||
#Dilute the ON at least 1/100, into 20-25mL in | #Dilute the ON at least 1/100, into 20-25mL in a 200mL Erlenmeyer | ||
#Grow the diluted bacteria until they reach an OD600 between 0.2 and 0.5 | #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 | #Put the eppendorfs in ice, to make sure they are cold | ||
| Line 17: | Line 23: | ||
#Store the competent cells in a -80°C freezer. | #Store the competent cells in a -80°C freezer. | ||
[[UA Biophysics:Protocols|Return to Protocols]]<br> | |||
Latest revision as of 13:09, 29 June 2023
Material
- TSS Buffer
- Overnight (E.coli MG1655)
- LB 25mL in a 200mL Erlenmeyer
- Sterile epperdorfs
Protocol
- Grow an ON in LB media (the day before)
- Dilute the ON at least 1/100, into 20-25mL in a 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.
