Mike Poidinger skspoidn at susssys1.rdg.ac.uk
Fri Apr 10 08:08:21 EST 1992

In response to:

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Message-Id: <9204091841.AA13249 at genbank.bio.net>
From: falquet at ch.unige.sc2a
Date: 9 Apr 92 13:20:45 GMT
Status: R

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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alternatively, try pCR 1000 from Invitrogen. It contains a
blue/white pre-cut vector containing appropriate T overhangs
to allow for direct cloning of PCR product (usually having a 3' A

Never used it myself, but hear good things about it from
Iain McGill (I.mcgill at uk.ac.crc).

It does cost a bit though.

Mike Poidinger
Dept of Microbiology
University or Reading

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