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freezing competent cells without dry ice or liquid N

competent cells

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#1 Haveshi

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Posted 24 January 2014 - 12:39 PM

Hi,

 

I am at a new lab at a new Institution we are making electrocompetent cells but we do not have liquid nitrogen or dry ice. I read that chilling ethanol at -80 degrees could work for snap freezing cells. We will even have to prepare them in a microcentrifuge since we do not have a refrigerated centrifuge. sad.png

 

Has anyone tried freezing with ethanol at -80 C? Is there an alternative for the snap freezing?

 

I would really appreciate your help.

 

Thanks

 

HS


Edited by Haveshi, 24 January 2014 - 12:41 PM.


#2 rkay447

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Posted 24 January 2014 - 01:24 PM

Keeping cells cold is one of the most critical aspects to making electrocompetent cells.  For your centrifuge, either move it into the cold room or chill the roter between spins.  Without dry ice or liquid nitrogen, -80 ethanol is pretty much your best option.  Aliquot the cells in the smallest volume possible (into pre-chilled tubes) so the cells freeze as quickly as possible.  It's certainly not ideal and chances are you won't get the absolute best cells but you should be able to make some that work ok.



#3 phage434

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Posted 24 January 2014 - 05:24 PM

I think cold ethanol will work fine. Watch your fingers. I think quick freezing is less important than most people believe. You can freeze/thaw competent commercial cells and lose only about a factor of two in competence (try it yourself!). You can probably do pretty well keeping cells on ice, and then just freezing them in the -80.



#4 Haveshi

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Posted 27 January 2014 - 10:44 AM

Thank you! That worked just fine! :)







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