Ligation Help Requested
hamel at ccu.umanitoba.ca
Tue Mar 30 13:25:02 EST 1993
Have you attempted to deproteinate (remove polymerase) your pcr
product DNA by treatment with proteinase K in 0.1% to 0.5% SDS (at
37oC to 55oC for 1/2 hour), followed by couple of ext'ns with phenol (Tris,
pH 8 sat'd)/ chloroform-2%n butanol, etc? If SDS still persists at
interface, further ext'ns needed in addition to ethanol ppt'n in presence
of 0.2 to 0.3 M NaCl with 2- 4 times volume ethanol (glycogen carrier,
about 1- 10 ug can be helpful).
Manitoba Vet. Virol.
email: hamel at ccu.umanitoba.ca
In article <27MAR93.19921652.0075 at UNBVM1.CSD.UNB.CA> E85J000 <E85J at UNB.CA> writes:
>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,
> (EMAIL: E85J at UNB.CA)
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