I'm still having problems with the E. coli cells that contain the DNA construct for the advanced tweezers experiments. The cells grew OK overnight on an LB+Amp50 agar plate but poorly in what I later realized was LB+Amp200 broth (too much ampicillin). I did a plasmid prep from these cells but got little or no plasmid (a faint smear only). Yesterday the postdoc inoculated another colony for me, into LB+Amp50, but this also stopped growing at a low density, and a plasmid prep gave no plasmid at all.
The E. coli cells are strain DH5alpha, which should be quite vigorous, and the plasmid is a derivative of pGEM and so should have a high copy number, even with a 12 kb insert. Perhaps the cells aren't what they're supposed to be. Maybe there's something wrong with my medium (and the postdoc's medium). Perhaps there was something wrong with my original LB+Amp plate. Perhaps the plasmid replicates poorly because it's so big. Perhaps the plasmid is present in the cells but lost during purification, because the miniprep kit doesn't give good recovery for DNAs bigger than ~10 kb.
Update: this morning i inoculated colonies from the LB+Amp plate into plain LB and LB+Amp50. The cells in plain LB seem to be growing faster, so maybe the plasmid is toxic. I'd better go back and reread the thesis chapter of the M.Sc. student who made it.
As I recall, the plasmid itself was replicated poorly, but the cells grew as regular DH5alpha E. coli did. My yields from maxipreps were always lower than for short plasmids, but I can't remember any problems with cell growth.
ReplyDeleteThanks, I was hoping you might remember. The cells grew a bit slower in LB+Amp, but growth of both cultures flattened out early, at about OD=0.5. SO I suspect there's something wrong with the medium.
ReplyDeleteLet me know if there's anything I can look up from this end (lab notebooks, duplicate stocks, etc.)
ReplyDeleteLet me know if there's anything I can look up from this end (lab notebooks, duplicate stocks, etc.)
ReplyDelete