Knight:TOPO TA cloning

Purpose
Efficient cloning of PCR products.

Materials

 * PCR product generated with Taq polymerase (for 3' A tails)
 * TOPO cloning kit

Reshma's procedure
(Note that the kit details a slightly different procedure which may give superior efficiencies.)
 * 1) Mix 1 &mu;L PCR product (amplified with Platinum PCR supermix) with 0.5 or 1&mu;L TOPO vector (0.5&mu;L should be sufficient.)
 * 2) Incubate 5 mins on the bench top
 * 3) Place on ice
 * 4) Transform

Resources

 * Website
 * Manual
 * Product notes
 * Brochure
 * pCR4-TOPO

Patents

 * 1) US Patent 6,916,632; Methods and reagents for molecular cloning. (On topoisomerase-mediated cloning using a 5' overhang and blunt end.)  [[media:Chesnut05.pdf|pdf]] html
 * 2) US Patent 5,766,891; Method for molecular cloning and polynucleotide synthesis using vaccinia topoisomerase [[media:Shuman98.pdf|pdf]] html
 * 3) US Patent 6,653,106; Topoisomerase based ligation and cloning methods [[media:Shuman03.pdf|pdf]]
 * 4) US Patent 6,548,277; Method for molecular cloning and polynucleotide synthesis using vaccinia topoisomerase [[media:Shuman03a.pdf|pdf]] html
 * 5) US Patent 5,487,993; Direct cloning of PCR amplified nucleic acids [[media:Herrnstadt96.pdf|pdf]] html
 * 6) US Patent 5,827,657; Direct cloning of PCR amplified nucleic acids [[media:Herrnstadt98.pdf|pdf]] html