Ligation Help Requested

E85J000 E85J at UNB.CA
Sat Mar 27 17:26:45 EST 1993


Dear peoples:

I'm in serious need of some help!  I have a PCR product with
engineered NdeI cut sites at both 5' & 3' termini.  I'm trying,
without any success, to cut and ligate into the NdeI site of
pET-11a.  When I started the project, the supplier of the vector
suggested that I have 10 bp flanking the cut sites "to ensure efficient
cutting".  After having invested in the primers and amplifying the
fragment, I discovered a paper in Biotechniques suggesting that the
cutting efficiency @ termini is really, really low...

My problem now is that i'm still unable to cut efficiently enough to
get ligation.  I've tried cutting with BSA carrier and Spermidine to
stabilize both the enzyme and DNA, with no luck so far...the only
transformants I seem to be able to get are the back-ground /
re-ligated plasmid that was not 100% phosphatased by CIP....

So, any suggestions on cutting my insert?

                thanks in advance,

                                  Pat FitzPatrick Biology Dept,
U.N.B. (Canada)

                                  (EMAIL: E85J at UNB.CA)



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