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

- good quality DNA (insert and vector). The vector tends to pick up the smallest molecule

- use correct cloning strain. Tandem array are unstable

- do not keep plasmids with tandem arrays in liquid culture in the cold room. If you keep then, freeze the cells.

- tandem arrays can get rearranged after being transformed back into cells, always check.

why is there a problem with Pstblue-1? Can you alter

-the growth conditions. Use a better media, bevelled flask, lower growth temperature
-host strain

to improve the quality of DNA? Poor vector DNA, just makes your life a little more difficult. I have never used Ale I and BBVCI , and so can not advise. Are these enzymes problematic?

Just don't over do the dephosphorylation. It can damage the ends of you vector. Is your T4 ligase okay. That enzyme easily goes off. Incubating the ligation at a lower temperature ie 16 C or 4 C sometimes helps.