GeneArt™ Site-Directed Mutagenesis System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Use 20–25 ng of the target plasmid per 50 µL of mutagenesis reaction as a starting point.	- 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

Upstream tips
- Use 20–25 ng of the target plasmid per 50 µL of mutagenesis reaction as a starting point.
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

Site-directed mutagenesis and cloning
The generation of FLAG-tagged and HA-tagged human wild-type SULT4A1 plasmids (pFLAG-SULT4A1-WT and pHM6-SULT4A1-WT, respectively) has been described previously . To generate the FLAG-tagged human variant SULT4A1 plasmid (pFLAG-SULT4A1-VAR), the same primers and procedure were used as in Mitchell et al except that the template for the PCR was cDNA from IMR-32 cells, which only express the variant SULT4A1 transcript. The pFLAG-SULT4A1-TV>AE and pFLAG-SULT4A1-KTV>QAE mutants were generated by the GENEART site-directed mutagenesis system (Life Technologies) using pFLAG-SULT4A1-WT as template and the following forward and reverse primers; FP-TV>AE, 5′-GGAAGGACA-TCTTCCCGTCCATGAATGAGAAG-3′, RP-TV>AE 5′-CTTCTCATTCATGG-ACGGGAAGATGTCCTTCC-3′, FP-KTV>QAE 5′-AGAGTTGGGCTGTGGA-GGACATCTTC-3′, RP-KTV>QAE , respectively. Clone sequences were verified by sequencing.



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Manufacturer protocol

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

Download manufacturer protocol

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