Cloning a PCR product
dkim at unm.edu
dkim at unm.edu
Mon Jun 27 11:00:42 EST 1994
(Omitted query about directionally cloning PCR products)
I remember that the Stratagene cDNA synthesis kit used a very clever idea
for directional cloning, in which 5-methyl-dCTP was used as one of the
precursors in the first strand reaction. This way, the internal XbaI sites
are protected and the sites in the primer are cut.
Could a variatio on this be used here? You'd have to do PCR with the methyl-
dNTP, then one last cycle with an excess of the regular dNTP. In this way,
internal sites would be hemimethylated, but the primers would ensure non-
methylated ends.
Daniel Kim
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