Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids - PubMed (original) (raw)

Comparative Study

. 1993 Feb 25;21(4):817-21.

doi: 10.1093/nar/21.4.817.

Affiliations

• PMID: 8451184
• PMCID: PMC309212
• DOI: 10.1093/nar/21.4.817

Free PMC article

Comparative Study

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

A C Boyd. Nucleic Acids Res. 1993.

Free PMC article

Abstract

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 recombinant molecules are efficiently excised from the ligation products by Cre recombinase acting on pairs of lox sites within directly repeated vector molecules flanking insert DNA. Recombinants are introduced into cells conventionally by transformation or electroporation. In both a model system and the cloning of PCR products, yields approaching those obtainable in cohesive-end cloning were achieved. Applications of the technique to cDNA library generation and recovery of DNA from archive material are discussed.

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References

1. 1. Nucleic Acids Res. 1986 Oct 10;14(19):7617-31 - PubMed
2. 1. Methods Enzymol. 1991;204:63-113 - PubMed
3. 1. Gene. 1987;51(1):69-75 - PubMed
4. 1. Methods Enzymol. 1987;152:94-110 - PubMed
5. 1. Nucleic Acids Res. 1988 Aug 11;16(15):7583-600 - PubMed

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