A-tailing Protocol for Blunt-end Fragments

Pfu DNA Polymerase(a)-generated blunt-end PCR fragments can be ligated into the pGEM®-T and pGEM®-T Easy Vectors(b,c) if the fragments are first A-tailed using Taq DNA Polymerase(a). 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.