Impossible PCR product cloning

Carl_JF_Simard at hotmail.com Carl_JF_Simard at hotmail.com
Fri Jun 9 13:34:35 EST 2006


Jose de las Heras a écrit :

> "arash arashkia" <aarashkia at yahoo.com> wrote in message
> news:mailman.85.1149358922.18505.methods at net.bio.net...
> >I am trying to clone in pcDNA3.1(+), a 100bp PCR product (BamHI/EcoRI
> >treated at the ends), after purification with PCR Product purification kit
> >(Roche). Although the yield decreases after purification, I can see my band
> >in gel electrophoresis.( 100bp is about in cut-off of the purification
> >kits). 5 times try to clone have failed. Has anyone an idea or protocol in
> >this regard?
> >
> >  B.R.
> >
> >  Arash
>
> For this sort of thing I tend to intermediate-clone into something like
> pGEM-TEasy. I add the restriction sites I want in the primers, amplify, and
> clone the PCR products directly into the pGEM vector (TA cloning). Then cut
> my fragment from there and clone as usual into the final vector.
>
> Jose

Same thing for me. With the added benefit that, once in pGEM-T easy,
you can sequence your amplified products to be sure no mutation occured
during the amplification.



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