title: Polymerase chain reaction-based gene removal from plasmids.
abstract: This data article contains supplementary figures and methods to the research article entitled, "Multiplex gene removal by two-step polymerase chain reactions" (Krishnamurthy et al., Anal. Biochem., 2015, doi:http://dx.doi.org/10.1016/j.ab.2015.03.033), which presents a restriction-enzyme free method to remove multiple DNA segments from plasmids. Restriction-free cloning methods have dramatically improved the flexibility and speed of genetic manipulation compared to conventional assays based on restriction enzyme digestion (Lale and Valla, 2014. DNA Cloning and Assembly Methods, vol. 1116). Here, we show the basic scheme and characterize the success rate for single and multiplex gene removal from plasmids. In addition, we optimize experimental conditions, including the amount of template, multiple primers mixing, and buffers for DpnI treatment, used in the one-pot reaction for multiplex gene removal.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States.
authors.forename: Vishnu Vardhan
authors.initials: VV
authors.lastname: Krishnamurthy
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States.
authors.forename: John S
authors.initials: JS
authors.lastname: Khamo
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States.
authors.forename: Ellen
authors.initials: E
authors.lastname: Cho
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States.
authors.forename: Cara
authors.initials: C
authors.lastname: Schornak
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
keywords: Gene removal; Plasmids; Polymerase chain reaction; Restriction-free cloning