Impossible PCR product cloning

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


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Did you add some additional bases to the primers that the restriction
enzymes have something to get hold on? Check the NEB catalog tech
section for details.

Additional things to do are:

dephosphorylate your vector (SAP)

make sure your PCR product is what you want (test by cutting,
sequencing)

if there is another enzyme cutting between your enzymes, do another cut
to reduce chance of self ligation and transfer the digest

do a blunt end (or TA overhang) cloning of your product first and then
cut it out and recone it.

Just some thoughts.

HTH, Wolfgang



arash arashkia wrote:

"arash arashkia" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ].n et...

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
--
Musha ring dum a doo dum a dah - [Only registered users see links. ]

Jose de las Heras a écrit :


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.