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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