sb500194z_si_001.pdf (916.27 kB)
Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly
journal contribution
posted on 2015-03-20, 00:00 authored by Hans Jasper Genee, Mads Tvillinggaard Bonde, Frederik
Otzen Bagger, Jakob
Berg Jespersen, Morten O. A. Sommer, Rasmus Wernersson, Lars Rønn OlsenUSER cloning is a fast and versatile
method for engineering of
plasmid DNA. We have developed a user friendly Web server tool that
automates the design of optimal PCR primers for several distinct USER
cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction
of virtually any type of site-directed mutagenesis by designing optimal
PCR primers for the desired genetic changes. To demonstrate the utility,
we designed primers for a simultaneous two-position site-directed
mutagenesis of green fluorescent protein (GFP) to yellow fluorescent
protein (YFP), which in a single step reaction resulted in a 94% cloning
efficiency. AMUSER also supports degenerate nucleotide primers, single
insert combinatorial assembly, and flexible parameters for PCR amplification.
AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/.