We have a cDNA expression library cloned into SCS1 E.coli, stored and used in 384 well plate format (as a Mastercopy and several daughter copies). For years, we could generate good copies (in new 384 well plates) of the daughter copies even with 6-8 cycles of thawing/freezing of the daughter copies. Now, after 2 times of thawing/freezing, the growth of the bacteria of the fresh copy is quite irregular across the plate - cells in the middle of the plate grow less than at the rim of the plate. We tried to "revive" them by plating them on agar plates and then inoculate new 384 well plates from these agar plates. Unfortunately, we still observe the same problem.

We use 2 YT medium, 2 % glucose, the right amount of ampicillin and canamycin plus 1x HMFM for freezing and long term storage.

Does anyone have experience with those cells and the medium used or suggestions? Since we're operating in 384 well plates and with 384 pin-gadgets for copying, the viscosity of the medium should be as low as possible, to avoid cross contamination.