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strange glycerol stocks - strange glycerol stocks (Apr/30/2002 )

I've had this wierd problem with some -80C glycerol stocks of XL-1 Blue cells with a pET28 vector + insert; when I streak the cells (kan-LB-agar) they form colonies and when I innoculate liquid cultures they grow to OD600 ~0.5-0.6 but then stop growing. I need to make minipreps, so I'd like an OD600 of 1.0. Has this ever happened to anyone, and if so what can be done?


Check the concentration of glycerol in your stocks. Expression hosts (DE3 lysogeny) containing constructs are unstable in glycerol above 8% v/v.


A typical problem. Grow the cells as before. When you reach the OD of 0.5, transfer 100 ul of culture to 100 ml of fresh antibiotic containing media. This time the OD will go up all the way to 1.4.

Good Luck

Sam Brilliant
Ph.D. Rescue Team
A division of DSTF-Global


I've done what you suggest. It simply doesn't work. DE3 lysogeny aren't stable at high conc of glycerol. This is straight from the horse's (Novagen) mouth.