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Problem with growing up from cell stocks - (Oct/10/2007 )

After I managed to successfully ligate my insert into my plasmid I transformed it into TOP10 cells and after checking the colony was correct I made a cell stock with glycerol and froze down. When I needed more DNA I just used the stock to streak a plate and picked colonies, however, recently every time I streak the plate (with Ampicillin) I get colonies but then when I incubate them in LB +Amp there is no growth. I am using the exact same cell stock as previously, what could have changed and how do I fix it?



have you tried to grow bacteria directly from the frozen stock?



Is your ampicillin okay? on your plates, its notorious for degrading so if you're plates are old or ampicillin stocks are old they may not work effectively (Carbenicillin is much more stable than ampicillin). Zek is right, try straight from the stock first if that doesn't work, then fresh ampicillin and check your top 10 cells for ampicillin resistence.

All the best

-lost in the lab-

Hi, thanks for replie, yes I have been trying to grow up from frozen stock and I have made new ampicillin plates and tested with other cell stocks I have and everything seems fine. It's really frustrating as they won't grow up but there didn't used to be a problem and I'm doing the exact same thing!

Could I transform some new cells with some DNA I have and grow them up again that way?