Freeze-Thaw competent cells - (Mar/15/2007 )

Hi,

I'm wondering if it is possible to re-freeze competent cells after thawing without great loss of transformation efficiency?
We have 100µl glycerol stocks of competent cells (CaCl2) and sometimes I only need 50µl. I'm wondering if I can put the remaining 50µl back into the -80° freezer.
Any ideas?

Thanks!

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I wouldn’t recommend it. They remained competent, but they’ll decrease competency dramatically. I have done that with commercial high competent cells, they still worked.
Why don’t you make stock of 50 and 100 ul next time you prepare competent cells.

If you just need 50 ul, I would use the 50 ul remained as a negative control.

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The commercial competent cells come with instructions, we have a small round container in the -80 full of isopropanol. You take the tube of competent cells and put into the -80 isopropanol for like 30 seconds (snap-freeze) then place in rack. Cells worked okay for me after this but did not do calculation to see if efficiency was lost.

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We lose about 3x transformation efficiency after 1 freeze/thaw and after several freeze thaws have about a total of 6x less transformation efficiency. It seems to remain at that level. This is with the CCMB80 competent cell preparation described here:
http://openwetware.org/wiki/TOP10_chemically_competent_cells

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We experienced loss in tranformation efficiency by about 50% after 1 freeze thaw.

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