TY - JOUR
AU - 
AB - Here we describe a straightforward, efficient, and reliable way to clone an insert of choice into a plasmid of choice without restriction endonucleases or T4 DNA ligase. Chimeric primers containing plasmid sequence at the 5′ ends and insert sequence at the 3′ ends were used to PCR- amplify insertion sequences of various sizes, namely the genes for GFP (gfp), β-D-glucuronidase (gusA), and β-galactosidase (lacZ), as well as the entire luxABCDE operon. These inserts were employed as mega-primers in a second PCR with a circular plasmid template. The original plasmid templates were then destroyed in restriction digests with DpnI, and the overlap extension PCR products were used to transform competent Escherichia coli cells. Phusion DNA polymerase was used for the amplification and fusion reactions, so both reactions were easy to monitor and optimize. Keywords overlap extension PCR cloning; recombinant vector; Phusion; restriction enzyme ligation independent Numerous alternative approaches to PCR cloning (1) have been developed, including TA cloning (2), ligation independent cloning (LIC) (3–4), recombinase-dependent cloning (5– 7), and PCR-mediated cloning (8–10). The practical utility of any cloning method is predicated upon its reliability, rather than its convenience, price, or efficiency under optimum conditions. The methods that are easiest to 
TI - Overlap Extension PCR Cloning: A Simple and Reliable Way to Create Recombinant Plasmids
JF - BioTechniques
DO - 10.2144/000113418
DA - 2010-06-01
UR - https://www.deepdyve.com/lp/unpaywall/overlap-extension-pcr-cloning-a-simple-and-reliable-way-to-create-HmrItZBLQj
DP - DeepDyve
ER -