Cloning PCR fragment into the expression vector
mdccsw at nus.edu.sg
Tue Mar 5 03:34:40 EST 2002
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.
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