Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

Making competent cell from freeze-thawed stock - (Jan/29/2010 )

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


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...


@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!