Cloning PCR fragment into the expression vector
nkuperw at yahoo.com
Thu Mar 7 16:36:34 EST 2002
Joyce <mdccsw at nus.edu.sg> wrote in message news:<20020305083440.30392.qmail at ww02.jatek.com>...
> Dear Colleagues,
> Need help. I am doing some blunt end ligation to clone a PCR product (~700
> bp) into an expression plasmid (~4.9 kb), but couldn't get it work. can
> anyone give any suggestion?
> For the PCR product, I have purify through agarose gel to get rid of Taq
> polymerase before doing ligation while the vector, a double digestion was
> performed with EcoRI and XhoI and Klenow fragment + dNTP was added to fill
> the 5' overhang. Then the 4.9 kb band was purified from agarose gel and
> performed dephosphorylation with Alkaline phosphotase to avoid self
> ligation. The linearised vector was again purified from agarose gel to get
> rid the Alkaline phosphatase (which will inhibit ligation). Ligation process
> was performed before transformation was done.
I think this has already been mentioned before, but if you're pcr'ing
your fragment, just engineer the sites into your primers and digest
your pcr reaction. No need for phosphatasing, fill-ins and all other
complicated steps. It might take a day or two to remaker/order
primers, but labor wise, I think it's a much easier path.
Also remember, the oligos you order/make for pcr are NOT
phosphorylated. If you dephosphorylate your vector and you don't
kinase your oligos, you will not be able to ligate your PCR fragment.
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