DNA-CLONING METHOD USING A CRE-LOX VECTOR UNDER CONDITIONS OF MACROMOLECULAR CROWDING

摘要

The method uses a plasmid vector, p91ox5, 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). Secondly, circular recombinant molecules are 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. Applications of the technique to cDNA library generation and recovery of DNA from archive material are discussed.