Cloning short DNA into plasmids by one‐step PCR

Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids.

The method uses a novel plasmid vector, p9lox5, containing a site-specific recombination sequence lox from the lox/Cre recombinase system of bacteriophage P1. There are two distinct stages. Firstly, vector and fragment DNAs are ligated intermolecularly under conditions of macromolecular crowding (15% polyethylene glycol 6000) which accelerate blunt-end joining a thousandfold. Secondly, circular...

Multiple Antibiotic Resistance Plasmids Allow Scalable,
PCR-Mediated DNA Manipulation and Near-Zero Background Cloning.

We have constructed two plasmids that can be used for cloning as templates for PCR- -based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418) and hygromycin B in Saccharomyces cerevisiae simultaneou...

Identification and cloning of integration site of DNA by PCR.

Gene walking by PCR amplification of short fragments from Taq DNA polymerase--modified P1 plasmid DNA and TA cloning.

1.Belgrader, P., W. Benett, D. Hadley, G. Long, R. Mariella, Jr., F. Milanovich, S. Nasarabadi, W. Nelson et al. 1998. Rapid pathogen detection using a microchip PCR array instrument. Clin. Chem. 44:2191-2194. 2.Belgrader, P., J.K. Smith, V.W. Weedn and M.A. Northrup. 1998. Rapid PCR identity testing using a battery-powered miniature thermal cycler. J. Forensic Sci. 43:315-319. 3.Belinda, A.J.G...

Cloning PCR products into T vectors.

MATERIALS Bacteriophage T4 DNA ligase T vector Target DNA (25 μg/ml), amplified by PCR When the PCR mixture contains more than one or two bands of amplified DNA, purify the target fragment by electrophoresis through low melting/gelling temperature agarose (please see Recovery of DNA from Low-meltingtemperature Agarose Gels: Organic Extraction). If not purified by gel electrophoresis, PCR-amplif...