One-step generation of error-prone PCR libraries using Gateway® technology.

ABSTRACT:

Background

Error-prone PCR (epPCR) libraries are one of the tools used in directed evolution. The Gateway® technology allows constructing epPCR libraries virtually devoid of any background (i.e., of insert-free plasmid), but requires two steps: the BP and the LR reactions and the associated E. coli cell transformations and plasmid purifications.

Results

We describe a method for making epPCR libraries in Gateway® plasmids using an LR reaction without intermediate BP reaction. We also describe a BP-free and LR-free sub-cloning method for in-frame transferring the coding sequence of selected clones from the plasmid used to screen the library to another one devoid of tag used for screening (such as the green fluorescent protein). We report preliminary results of a directed evolu

SUBMITTER: Gruet A 

PROVIDER: S-EPMC3349575 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature