Freezer culture fails to retain plasmid - (Oct/15/2004 )

In my lab we save strains of bacteria by freezing them in 60% glycerol at -80 degrees C. When we need the bacteria again, we scoop out some of the frozen stock and put it in fresh liquid media. I don't know how common this practice is, but it has always worked fine for us. I have an E coli strain with a plasmid in it I am trying to save by freezing down as usual, but upon revival it doesn't seem to have the plasmid which is the whole reason I am saving the strain. The wierd part is that I already put this strain in the freezer and named it #266. We always make a second and often a third freezer copy in case the first one is lost or runs out. Strain #266 can be revived and contains plasmid, but every time I use the revived broth to make a new freezer culture the plasmid is lost.
Any ideas?

Are you sure your antibiotics works?

Actually I am in the process of checking that now. I suspect the plate I originally grew them on had started to fade in terms of antibiotic. When I made a new antibiotic stock, no bacteria at all grows in it. I want to try growing overnights in a lower amount of the new antibiotic stock. That way can get some growth but hopefully still ensure the plasmid remains. I should be able to find out early next week if this solves my problem.

Is your plasmid toxic for the bacteria? Sometimes, if the plasmid has an insert toxic for the bacteria, the plasmid, or the insert, is loose after freezing.

Good luck