HIV-pCL-CG - problem w/ bacteria - (Aug/07/2008 )
I want to make a lentivirus (pCL-CG; http://www.addgene.org/pgvec1?f=c&cmd=...entifier=12157) containing rat CD2 extracellular domain and tetracycline responsive element (TRE). I had some problems w/ vector preparation (some smears occured) but I think (or maybe I'm wrong) that I have ligated rCD2-TRE into it. The bacteria grow on an amp plate but they die in liquid culture. I have tried to culture it in LB, terrific broth, with and w/o glucose and in 37 or 32oC for 10-16hrs (I have tried all of the combinations). But either bacteria die (clumps or "gum-like: appearance) or look more or less good but I can't extract plasmid from them.
I had similar problems w/ pMIG (MSCV-IRES-GFP) vector (from Addgene as well) - smears; but I could have ligated insert w/ wector. Now I can't grow bacteria with pCL-CG.
Did anybody have similar problems? Or maybe you could advise me something?
Thanks in advance!
I forgot to add that I have transformed DH5a and XL-1 blue with my ligation and with both I can't isolate plasmids despite that bacteria grow.