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.
Highlights
Experimental factors Plasmids were linearized by inverted PCR with a pair phosphorylated primers and recircularized by blunt-end ligation
A method based on two-step PCR was used to remove multiple gene segments from plasmids
The data provide a method and systematic characterization for removing one gene from plasmids based on inverted PCR and blunt-end ligation
Summary
The data are supplied with this article. The data provide a method and systematic characterization for removing one gene from plasmids based on inverted PCR and blunt-end ligation. The data provide the scheme and characterize the success rate for PCR-based multiplex gene removal. The data provide the optimization of methods, including the amount of parent template, multiple primers mixing, and buffer conditions for DpnI digestion, to achieve pure products with multiple genes removed from plasmids based on two rounds of PCR
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