Problems amplifying unstable construct in E.coli - Tips needed (Dec/13/2007 )
A collaborator gave me a plasmid with a gene of interest, and I need to amplify it. I was told by the person that this construct tends to be unstable, thus to limit recombination they grow bacteria at 30C, not at 37C. I transformed DH5alpha E.coli with this plasmid and saw decent size colonies after 24h at 30C. The problem is that when I inoculate an overnight liquid culture for midi prep, the bugs don't grow very well if at all. I do see some growth, but it neither smells like E.coli (does it smell differently when grown at 30C?), nor do I get enough plasmid by midi prep (I believe I do get some, but like 100x less than expected). What is happening - is the liquid culture overgrown with something else (why does it smell differently)? Should I use a different strain of E.coli for amplification of this construct? Should I grow it much longer than overnight? Any other suggestions?
I would use another strain. I also had problems with an unstable plasmid, I switched to the JM109 strain (because of recA- and LacIq) and it worked very well. Only JM109 does still has endonucleases. Maybe you can also use another strain, but I could not work with a strain with Tn5 and this was the only RecA-/LacIq strain without Tn5 in the house.
By the way, I had exactly the same problems with the liquid cultures.
Is there any chance that your new plasmid can express protein? If so, is the potential construct toxic?
You might be getting leaky expression, which could be caused by trace levels of lactose in casein-based media. If so, try adding some glucose to the media, as per Studier's 2005 paper (Protein Expr Purif. 2005 May;41(1):207-34.)