Sterility issues with plasmid reconstitution buffer? - (Apr/28/2014 )

In the final step of a plasmid prep after drying off the 70% ethanol used for washing, would it matter if I used non-sterile water (DNAse & RNAse free) or TE buffer? These buffers are used in non-sterile conditions in the molecular lab. 

I plan to use the prepared plasmid for tissue culture transfection which requires everything to be sterile.  What are the risks of introducing contamination at this step (e.g. bacterial, mycoplasma and yeast) into my cell cultures?

The risks are minimal - EDTA will kill off most things fairly effectively.

If you are worried you can add a drop of chloroform, shake and let sit.  This will kill off the bacteria etc by dissolving the membranes.

bob1 on Tue Apr 29 07:48:05 2014 said:

The risks are minimal - EDTA will kill off most things fairly effectively.

 

If you are worried you can add a drop of chloroform, shake and let sit.  This will kill off the bacteria etc by dissolving the membranes.

I normally reconstitute my plasmid DNA in water since I always verify the enzyme cut sites which require a EDTA-free buffer.  Would the use of water instead of TE buffer change reduced risk you eluded to?

Yes, potentially things can grow in water that wouldn't be able to grow in TE, but if you are using fresh milliQ water then there shouldn't be too much of a problem.