UA Biophysics:Protocols:Competent Cells: Difference between revisions

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==COMPETENT CELLS PROTOCOL==
==COMPETENT CELLS PROTOCOL==


#1.Grow an ON in LB media (the day before)
#Grow an ON in LB media (the day before)
#2.Dilute the ON at least 1/100, into 20-25mL in an 200mL Erlenmeyer
#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
#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
#Put the eppendorfs in ice, to make sure they are cold
#5.Check that the TSS Buffer is also cold
#Check that the TSS Buffer is also cold
#6.Take the culture and separate it into two 50mL falcon tubes.
#Take the culture and separate it into two 50mL falcon tubes.
#7.Leave the falcon tubes on ice during 10 min.
#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.


''**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.


 
*'''[[User:Sofia Alfonso-Sanchez|Sofia Alfonso-Sanchez]] ''':
 
*'''[[User:Sofia Alfonso-Sanchez|Sofia Alfonso-Sanchez]] 00:28, 1 August 2015 (EDT)''':

Revision as of 21:40, 31 July 2015

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 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.


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