GeneArt™ Site-Directed Mutagenesis PLUS System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Create a fresh dilution of 25X SAM from the kit-supplied 200X SAM in sterile, distilled water each time you perform the mutagenesis procedure - 25X SAM is not stable, and loses activity within a few hours after preparation

Protocol tips
- Create a fresh dilution of 25X SAM from the kit-supplied 200X SAM in sterile, distilled water each time you perform the mutagenesis procedure - 25X SAM is not stable, and loses activity within a few hours after preparation

Publication protocol

PCR, Sanger sequencing, cloning, and exome sequencing
PCR (42 cycles of 10 seconds at 94°C, 15 seconds at 55°C, and 1 minute at 68°C) was performed with Platinum PCR supermix (Life Technologies) and the primers 5′-GACTCTCTTGCATTAGGG-3′ and 5′-TGCAAAGTCAGCAGGAAATG-3′. PCR products were sequenced with the following primers: 5′-GGGAAGAGTGAAGAAGAAG-3′ and 5′-CATCGGGGATTGTTCAGATT-3′. The pReceiver-M43 plasmid encoding wild-type (WT) PIK3CB (NP_006210.1) was from GeneCopoeia. The D1067Y, D1067A, D1067V, and K805R mutants were generated by the GeneArt Site-Directed Mutagenesis PLUS System (Life Technologies). For generation of Rat-2–stable cell lines, the sequence corresponding to PIK3CB WT or D1067Y was inserted into pEF1/Myc-His vector by GenScript. Exome sequencing of parental and resistant cell lines was done as described previously, and the prevalence of PIK3CB mutations was determined using exome sequencing data from a panel of cancers downloaded from The Cancer Genome Atlas (TCGA) and processed as described previously (31, 32). Sequencing data were filtered for nonsynonymous coding variants not present in dbSNP and present at >20% allele frequency in the resistant pool only.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for GeneArt™ Site-Directed Mutagenesis PLUS System below.

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