Journal: Journal of Biological Engineering
Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Figure Lengend Snippet: Sequence elements facilitating the subcloning procedure . (A) A double stranded oligonucleotide was cloned into pGEM-T easy to provide two key elements flanking a BglII site. We inserted a 1166 bp PCR amplified fragment into this restriction site. In future applications this step may as well be achieved by TA cloning or other methods. (B) A key element consists of a Esp3I recognition site and a restriction site generating a NheI compatible overhang, that is not recognized by NheI. (C) We used NheI as target recognition site in the destination vector.
Article Snippet: Method In an example experiment we subcloned a 1166 bp long DNA fragment* from an entry vector (modified pGEM-T easy, Promega) into a NheI site of a destination vector (pEGFP-N1, Clontech).
Techniques: Sequencing, Subcloning, Clone Assay, Polymerase Chain Reaction, Amplification, TA Cloning, Plasmid Preparation