Cloning hell - (Dec/17/2008 )

Problem- Trying to clone a series of direct repeats (24x MS2 sites, ~1.5 kb) cut with Ale I and BBVCI into a similarly digested vector (PstBlue1, ~18kb). I've used Stbl2 and Sure II cells (followed transformation protocols provided by manufacturer) with no success. Any tips for cloning direct repeats??? This truly sucks.

Basic cloning strategy

1) Gel purify AleI/BBVCI MS2 fragments (Should mention that the MS2 sites are digested out of Pstblue-1, this construct gives really crappy mini/midi prep yields, probably sick)

2) Digest Pstblue1 vector with Ale I/BBVCI, then depho4 with antartic phosphatase

3) Ligate 1 and 2 (I've used a range of molar ratios, 1:3, 1:50, 1:100)

4) Transform with Stbl2 or Sure II, some colonies-usually parent vector religation

Thanks,

Squishy

hey squishy
repeats are awful
your parent re-ligation - is it the right size or are the bugs chucking out bits? - if they are not the right size, this has happened to me before and growing the bugs at 30 degrees post-transformation recovery instead of 37 has worked for me....have also plated and left at room temp for a few days instead of 37C....
good luck!