CLONING AND ANALYSIS OF PCR-GENERATED DNA FRAGMENTS

Methods are presented for the improved yield and analysis of blunt-end ed cloning of PCR-generated DNA fragments. We show that Ma DNA polymer ase polishing of Tag DNA polymerase-generated fragments increases the yield and efficiency of cloning. Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-spec ific primer, both the presence and orientation of cloned inserts can b e determined. Application of these methods allows the generation and c loning of a fragment in 1 day and the analysis of putative clones the next, thereby saving a substantial amount of both time and effort.