Preparing chemically competent cells

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(Materials)
Line 9: Line 9:
*5ml pipete
*5ml pipete
*200ml conical flask
*200ml conical flask
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*Culture tube
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*Repeating pipettor & tip that can make 100μl aliquots.
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*Repeating pipettor, tip that can make 100μl aliquots.
+
==Preparation==
==Preparation==
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#Grow a 5ml overnight culture of cells in LB media using a culture tube.  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.
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#Grow a 5ml [[Bacterial cell culture|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.
-
#Grow the 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).
#Split the culture into two 50ml falcon tubes.
#Split the culture into two 50ml falcon tubes.
'''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.
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#Remove supernatant.  The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful.
+
#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.
#Using a repeating pipettor, add 100 &mu;l aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.
#Using a repeating pipettor, add 100 &mu;l aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.

Revision as of 13:13, 16 January 2006

Contents

Materials

  • Plate of cells to be made competent
  • TSS buffer
  • LB media
  • Ice

Glassware & Equipment

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

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.

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. Using a repeating pipettor, add 100 μl aliquots to your chilled eppendorfs and store at − 80oC.

Buffers

TSS (50 mL)

  • 5g PEG 8000
  • 1.5 mL 1M MgCl2
  • 2.5 mL DMSO
  • LB to 50 mL

Filter sterilize (0.22 μm filter) and store at 4˚C or -20˚C.

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