Preparing chemically competent cells

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#Grow the diluted culture to an OD<sub>600</sub> of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD<sub>600</sub> 0.2)
#Grow the diluted culture to an OD<sub>600</sub> of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD<sub>600</sub> 0.2)
#Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later.  If your culture is Xml, you will need X tubes.  At this point you should also make sure that your [[TSS]] is being chilled (it should be stored at 4C but if you have just made it fresh then put it in an ice bath).
#Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later.  If your culture is Xml, you will need X tubes.  At this point you should also make sure that your [[TSS]] is being chilled (it should be stored at 4C but if you have just made it fresh then put it in an ice bath).
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#Split the culture into two 50ml falcon tubes.
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#Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.
'''All subsequent steps should be carried out at <math>4^o</math>C and the cells should be kept on ice wherever possible'''
'''All subsequent steps should be carried out at <math>4^o</math>C and the cells should be kept on ice wherever possible'''
#Centrifuge for 10 minutes at 3000 rpm and <math>4^o</math>C.
#Centrifuge for 10 minutes at 3000 rpm and <math>4^o</math>C.
#Remove supernatant.  The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful.  Pipette out any remaining media.
#Remove supernatant.  The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful.  Pipette out any remaining media.
#Resuspend in chilled [[TSS]] buffer.  The volume of [[TSS]] to use is 10% of the culture volume that you spun down.
#Resuspend in chilled [[TSS]] buffer.  The volume of [[TSS]] to use is 10% of the culture volume that you spun down.
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#Using a repeating pipettor, add 100 &mu;l aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.
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#Add 100 &mu;l aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.
==Related topics & References==
==Related topics & References==

Revision as of 10:30, 17 January 2006

Contents

Materials

Glassware & Equipment

  • Falcon tubes
  • 500μl Eppendorf tubes, on ice
  • 200ml conical flask
  • Repeating pipettor & tip that can make 100μl aliquots.
  • 5ml pipette

Preparation

  1. Grow a 5ml overnight culture of cells in LB media. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/500.
  2. Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)
  3. Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is Xml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4C but if you have just made it fresh then put it in an ice bath).
  4. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4oC and the cells should be kept on ice wherever possible

  1. Centrifuge for 10 minutes at 3000 rpm and 4oC.
  2. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
  3. Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down.
  4. Add 100 μl aliquots to your chilled eppendorfs and store at − 80oC.

Related topics & References

Based on a protocol from Kathleen McGinness, annotated by Josh Michener & Barry Canton. Original protocol published by Chung et al.[1]

  1. Chung CT, Niemela SL, and Miller RH. . pmid:2648393. PubMed HubMed [chung]
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