Marc Cox mcox at
Fri Aug 4 23:59:24 EST 2000

If you are going to dephosphorylate the pcDNA3 then you need to
phosphorylate the cloned insert.  However, it sounds like you are cloning
restriction sites onto the end of the insert.  In this case, after you cut
the insert with Hind III and XBA I, the ends will then be phosphorylated.

T4 polynucleotide kinase comes with a reaction buffer from most companies
like Promega or NEB.  The reaction conditions should be in the catalogue and
the ATP is already in the buffer.

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