Problem with cloning unstable HIV insert - (Jun/18/2003 )

I've been having trouble inserting the HIV env gene (almost 3kb) into an HIV molecular clone. I've tried a variety of vector:insert ratios, growth at 30 versus 37 degrees, and transformation into either Invitrogen's Top10 cells or their Max Efficiency Stbl2 cells. Both the vector and inserts are clean (digested with Xba I and Xma I), and a gel of the ligation reaction shows a band of the expected product, as well as bands of other ligation products. I get <5 colonies per transformation, and after growing them up for a mini-prep, the size of the construct is always much smaller than expected (smaller than the original 9kb-ish vector).

However, I have had little trouble expanding the original molecular clone, so perhaps instability is not a problem after all ... ?

In any case, does anyone have ideas on how to resolve this?

Hi Codex

just wanting to know if you managed to solve your env cloning. The lab I'm in we're trying to clone a novel env gene (approx 3kb), generated from RT-PCR from patient sera, into pCRII. Will get colonies but always there are deletions of insert or no insert??? We're about to give this cloning up as cannot think of what to try next. Have tried 30oC, lower AMP concentrations, HB101s/DH5a's/TOPO kit cells, to no avail.

Any help would be hugely appreciated!

Thanks
Jane