Salvaging a plasmid from a cell culture stored at the incorrect temperature

Question

I have E. coli with transformed plasmid on agar in a vial. It has been stored at -20 degree C without glycerol stock for 18 hours.

This is a continuation of: Survival of E. coli on agar plate at -20 degree C without glycerol stock

I have transfered this vial from -20 degrees to 4 degrees after 18 hours when I determined that I had made a mistake. Since this is a very important vial and I don't have another one, what should I do in this stage?

What is the procedure for saving the plasmid if I am unable to save the whole cell?

Answer 1

I would rinse the cells off the agar using a small volume of LB or similar rich medium. Then use a small aliquot (10 μl) to inoculate a 5 ml culture under selection to see if anything will grow. Meanwhile the remainder of the cell suspension from the vial can be used as input for your favourite miniprep procedure.

If necessary, the DNA that you recover should then be used to transform a suitable E. coli strain. Don't bother trying to analyse the rescued DNA first since polysaccharide contaminants from the agar will probably inhibit any restriction enzyme that you might use.

Good luck!