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Cloning Blunt-End Pfu DNA
Polymerase-Generated PCR Fragments into pGEM®-T Vector Systems
By Kimberly Knoche, Ph.D., and Dan Kephart, Ph.D.
Promega Corporation
Pfu DNA Polymerase-generated
blunt-end PCR fragments can be ligated into the pGEM®-T and pGEM®-T
Easy Vectors if the fragments
are first A-tailed using Taq DNA Polymerase.
We describe a method for A-tailing these PCR fragments and demonstrate its utility by
cloning two different Pfu DNA Polymerase-generated fragments into the pGEM®-T
Easy Vector, using the new 2X Rapid Ligation Buffer. We found that, depending upon the
size and yield of the amplified fragment, the tailing protocol must be adjusted to
optimize the insert:vector ratio for ligation. With some inserts we found that the
alpha-peptide
based blue/white screening results in both white and pale blue recombinant colonies. One
PCR product tested here gives such a mixture of recombinant colonies. With this particular
PCR fragment we found that the two different colony colors resulted from opposite
orientations of the insert.
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