Alanine substitution mutations were introduced into pSS607 with a Quikchange Lightning site-directed mutagenesis kit (Life Technologies). Mutant primers were designed with a genomics program provided by Agilent Technologies. The primers were purchased from integrated DNA technologies. The mutant plasmids were introduced into XL-Gold E. coli by transformation, as described in the Quikchange instruction manual. Plasmid DNA was extracted from the transformants with an IBI miniprep kit and sequenced commercially to confirm the presence of the mutation in the plasmid. The mutant plasmid DNA was introduced into R-1 with a Sigma–Aldrich yeast transformation kit. Genetic testing confirmed that the construct was correctly inserted, as described by Ananthaswamy et al. (5 (link)).
Quikchange lightning site directed mutagenesis kit
Manufactured by Thermo Fisher Scientific
The Quikchange Lightning site-directed mutagenesis kit is a tool used for introducing site-specific mutations into double-stranded plasmid DNA. It utilizes a proprietary enzyme blend and optimized reaction components to enable fast and efficient mutagenesis.
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2 protocols using quikchange lightning site directed mutagenesis kit
1
Introducing Alanine Mutations in pSS607
2
Site-directed mutagenesis of plasmid DNA
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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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