Impossible PCR product cloning

[Jose de las Heras]

"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
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