UA Biophysics:Protocols:Competent Cells: Difference between revisions

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(New page: 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 OD60...)
 
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COMPETENT CELLS PROTOCOL
==Material==
* [https://openwetware.org/wiki/TSS  TSS Buffer ]
* Overnight (E.coli MG1655)
* LB 25mL in a 200mL Erlenmeyer
* Sterile epperdorfs


1.Grow an ON in LB media (the day before)
==Protocol==
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**''
#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.


8.Centrifuge during 10 min at 3000rmp and 4°C
[[UA Biophysics:Protocols|Return to Protocols]]<br>
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.

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

  1. Grow an ON in LB media (the day before)
  2. Dilute the ON at least 1/100, into 20-25mL in a 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 following 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.

Return to Protocols

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