Prolonged Final Extension Time Increases Cloning Efficiency of PCR Products

Directional cloning of PCR products.

INTRODUCTION This protocol is for directional blunt-end cloning of DNA fragments. The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA. The products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction (Liu and Schwartz 1992) to re...

Blunt-end Cloning of PCR Products.

INTRODUCTION Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves. In almost all cases, ...

Rapid and reliable cloning of PCR products.

Hetero-stagger cloning: efficient and rapid cloning of PCR products.

A variety of methods have been developed for cloning PCR products, including blunt-end cloning (1), restriction cut back (2), ligation-independent cloning (3), uracil DNA–glycosylase (UDG) treatment of uracil-containing deoxyoligonucleotide primers (4,5) and TA cloning (6–8). Blunt-end cloning of PCR products often requires treatment of PCR products to polish the ends (9). Even with treatments,...

Polishing with T4 or Pfu polymerase increases the efficiency of cloning of PCR fragments.

Several of the polymerases widely used for PCR, such as Taq DNA polymerase, have been found to exhibit terminal deoxynucleotide transferase activity (1, 2, 3). This terminal transferase activity, for the most part is limited to the addition of a single nucleotide and has been named 'extendase' activity. In a widely cited study, Clark has shown that when the 3' base is a cytosine nucleotide, the...