falquet at sc2a.unige.ch falquet at sc2a.unige.ch
Thu Apr 9 08:20:45 EST 1992

In article <9204010949.AA16192 at genbank.bio.net>, UDHA094 at OAK.CC.KCL.AC.UK (MARCEL KUIPER) writes:
> Dear colleagues,
> I am trying to introduce restriction sites at the 5' and 3' end of PCR
> products, to ligate the digested product in a vector. Until now I've
> been unsuccesfull in doing so. At the 5' end I've introduced a KpnI site
> with 6 bp in front of it; at the 3' end I've introduced a BglII site with
> 2 bp extra at the 3' end (should be sufficient according to the New England
> Catalog).
> After PCR, I treat the products with Proteinase K to remove Taq polymerase
> sticking to the ends, and digest the product with the restriction enzymes
> and ligate it into a vector digested with KpnI and BglII.
> Unfortunately, the transformed ligation product doesn't yield any colonies.
> Can someone help me solve this problem?
> Thanks very much in advance,
> Marcel Kuiper.

It is always difficult to cut close to the ends of a PCR product.

- Kinase the ends (Oligos for PCR don't have 5' phosphates)
- Blunt end your PCR products with Klenow 
- Blunt ligate into a blunt cutted/cipped vector
- Transform, miniprep, digest with KpnI and BglII (this time they cut fine)
- Low melt purify your fragment 
- Ligate into the final vector between KpnI & BglII sites.

It is longer but it works...

Laurent FALQUET and Eduardo GONZALEZ (U.K. Laemmli group)

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