2 replies to this topic

[hogthehedge]

Hi everyone,

I recently made a batch of competent dh5alpha E. coli using a transformation storage buffer containing the following:
PEG (MW 8000), MgSO4, DMSO and LB.
I realized afterwards that I had added five times the amount of MgSO4 specified in the protocol.
Has anyone ever made a similar mistake? Do you think my cells will still work?
Can anyone explain what each of the ingredients is supposed to do in this buffer?

Thanks in advance for your help!

[bob1]

The salts are usually the component that keep the cells competent.  I suspect that you will be able to transform the cells, but that it will be low efficiency.  You should test an aliquot of cells each time you prepare them to ensure that you have good transformation efficiency with a standard plasmid.  This way you can eliminate one more potential error if your experiments don't work.

[hogthehedge]

Thanks, Bob. I actually think it's 2.5 times the MgSO4, not 5. I'll try an aliquot and check.
Also thanks for your other reply on the unidentifiable contaminant. You were right, it turned out to just be lots of debris and gunk from old unfiltered serum. Not sure what is lost by filtering serum, but the cultures look a lot cleaner now.