Cloning and analysis of PCR-generated DNA fragments.
Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments. We show that Pfu DNA polymerase polishing of Taq 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-specific primer, both the presence and orientation of cloned inserts can be determined. Application of these methods allows the generation and cloning of a fragment in 1 day and the analysis of putative clones the next, thereby saving a substantial amount of both time and effort.
Costa
G L
GL
Stratagene Cloning Systems, La Jolla, California 92037.
Grafsky
A
A
Weiner
M P
MP
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004247:DNA