Hetero-Stagger Cloning: Efficient and Rapid 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,...

Rapid and reliable cloning 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...

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5' end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3' overhangs designed to specifically complement each other. The combin...

Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase.

By incorporating dUMP residues into the 5' end of PCR primers, one can generate products which, after treatment with uracil DNA glycosylase (UDG), contain 3' overhangs. These overhangs can be annealed to vector molecules with complementary overhangs generated in a similar fashion and transformed directly into Escherichia coli without the need for ligase. We have tested this method of ligation-i...