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Making competent cell from freeze-thawed stock


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#1 Brertemy

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Posted 29 January 2010 - 02:21 PM

Hi, I thawed a frozen stock of competent E-coli cells from -70 and then freezed it again. My question is, will I be able to make competent cells if I streak this refrozen cells on plate, get a single colony, and make overnight culture and do the whole Cacl2 treatment?
This is my last stock of these E-coli cells... I was wondering if i can salvage them
-brertemy

#2 HomeBrew

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Posted 29 January 2010 - 02:30 PM

Hi Brertemy -- welcome to the BioForums!

You'll be fine -- we routinely start a new plate from one of the final tubes in our stock of competent E. coli and use it to prepare a new batch. We use the RbCl2 method rather than the CaCl2 method -- does your method incorporate glycerol as a cryoprotectant? You'll need to have something to protect the cells if you're going to freeze your new batch...

#3 Brertemy

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Posted 29 January 2010 - 02:35 PM

@homebrew tnx for the reply.
The thing is, my final stock was subjected to one freeze thaw cycle, so I was wondering if this would have a drastic effect on the competency of this strain...

I am using the protocol with the the CaCl2, and PIPES/glycerol buffer..So yes I do have a cryoprotectant for freezing it..
Tnx a lot!
-brertemy




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