3 replies to this topic

[FruitflyD]

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?

[sage]

Are you sure your antibiotics works?

[FruitflyD]

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.

[heigor]

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