Cloning PCR fragment into the expression vector

Nicolas Kuperwasser 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.

Good luck

NK




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