Site-directed mutagenesis of plasmid DNA

We introduced the nucleotide substitutions into pSS607 with a Quikchange Lightning site-directed mutagenesis kit (Life Technologies). We designed mutant primers with a genomics program provided by Agilent Technologies. The mutant plasmids were introduced into XL-Gold E. coli by transformation, as described in the Quikchange instruction manual. We extracted plasmid DNA from the transformants with an IBI miniprep kit (MIDSCI) and had it sequenced commercially to confirm the presence of the mutation in the plasmid (SeqWright). We introduced the mutant plasmid DNA into R-1 with a Sigma Aldrich yeast transformation kit. Genetic testing described elsewhere (Ananthaswamy et al. 2010 (link)) confirmed that the construct was correctly inserted.

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Rahman H., Rudrow A., Carneglia J., Joly S.S., Nicotera D., Naldrett M., Choy J., Ambudkar S.V, & Golin J. (2019). Nonsynonymous Mutations in Linker-2 of the Pdr5 Multidrug Transporter Identify a New RNA Stability Element. G3: Genes|Genomes|Genetics, 10(1), 357-369.

Publication 2019

Quikchange Lightning site-directed mutagenesis kit yeast transformation kit

E coli Gold Mutation Nucleotide Plasmid Plasmid r Primers Site directed mutagenesis Yeast

Corresponding Organization : Catholic University of America

Other organizations : University of Nebraska–Lincoln, Center for Cancer Research

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Variable analysis

independent variables

Nucleotide substitutions introduced into pSS607

dependent variables

Presence of the mutation in the plasmid

control variables

Quikchange Lightning site-directed mutagenesis kit (Life Technologies) used for introducing the nucleotide substitutions
Genomics program provided by Agilent Technologies used for designing mutant primers
XL-Gold E. coli used for transformation of the mutant plasmids
IBI miniprep kit (MIDSCI) used for extracting plasmid DNA from the transformants
Sigma Aldrich yeast transformation kit used for introducing the mutant plasmid DNA into R-1
Genetic testing described in Ananthaswamy et al. 2010 used to confirm the correct insertion of the construct

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