Lidstrom:Competent Cell Preparation

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(Chemically Competent E. Coli)
(Chemically Competent E. Coli)
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*You need fresh cells.
*You need fresh cells.
**Often people inoculate a few mL of the culture for overnight growth, then use 200 uL to inoculate the ~50 mL of culture they will make competent.  
**Often people inoculate a few mL of the culture for overnight growth, then use 200 uL to inoculate the ~50 mL of culture they will make competent.  
-
**Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old.  Other people (Mila) are mostly concerned about the OD being right at the time of harvest.
+
**Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old.  Other people (Mila) are mostly concerned about the OD being right at the time of harvest.
*You need to flash freeze the cells at the end of the procedure.  You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.
*You need to flash freeze the cells at the end of the procedure.  You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.
==ElectroCompetent E. Coli==
==ElectroCompetent E. Coli==
*This is not done frequently in our lab.
*This is not done frequently in our lab.

Revision as of 17:35, 10 April 2012

Back to Protocols


Chemically Competent E. Coli

Notes:

  • You need fresh cells.
    • Often people inoculate a few mL of the culture for overnight growth, then use 200 uL to inoculate the ~50 mL of culture they will make competent.
    • Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old. Other people (Mila) are mostly concerned about the OD being right at the time of harvest.
  • You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.

ElectroCompetent E. Coli

  • This is not done frequently in our lab.
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