double enzyme cut and ligation - (Sep/15/2005 )

Hello,

Now I try to clone my DNA fragment (1.4kb) in pUC plasmid with specific promoter(12.4kb). I cut both of them using AgeI and XhoI. For my DNA fagment, I can get my expected band. pUC vector seems to be cut not completely because I directly transform the digested pUC vector to E coli cells, I still can get a lot of colonies. Please note xhoI and age I site is too close in my pUC vector.
The following are what I have done:
CIP to dephosphorylate,
gel purify,
cut vector separately,
all of them seems to be useless.
Please give me some sugestion!

Thanks a lot!

I guess your insert (12.4kb?) is too long for pUC19. It's really hard to clone DNA longer than 10kb in plasmid vectors, even it's possible.

Try phagemid or cosmid vectors.

And how far is it between AgeI and Xho I? Are the restriction site overlapped?

Good luck!

Too close as in overlapping cutting sites?

In that case you won't ever get both enyzmes to work properly.

If it's simply very close but not overlapping, then you have to consider that both enzymes need a certain number of nucleotides of overhang from their recognition site to work efficiently (see NEB catalogue or website for how many exactly).

To avoid this problem you could cut your vector with one of the enzymes, clone in some other bit of DNA and then use both enzymes, as long as you make sure that both cutting sites are still there.

I also seem to remember that Xho is blocked by dam methylation (but you'd better check that), so find out in which bacteria you've grown your vector and if they do this methylation.

LeserattePD

Thanks a lot!