Cell Culture Glycerol Stocks

[Denise]

Hi guys
there is this weird 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 inoculate liquid cultures they grow to OD600 ~0.5-0.6 but then stop growing. I need to make mini-preps, so I'd like an OD600 of 1.0. Has this ever happened to anyone, and if so what can be done?

[admin]

Hi there,
I would check the concentration of glycerol in your stocks. The expression hosts (DE3 lysogeny) containing constructs are unstable in glycerol above 8%

[kiki06]

This is actually a typical cell problem. I would grow the cells as before but 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.cheers