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

Mutagenesis of M23-AQP4
Both 4–6 amino acid serial alanine or glycine substitutions and point mutations were introduced into M23 AQP4 extracellular loops using the PCR-based GeneArt site-directed mutagenesis system (Invitrogen) (Table 1). Partially overlapping sense and antisense primers were designed from the M23 AQP4 sequence with the desired nucleotide mutation(s) located near the center of each primer sequence. Methylation and mutagenesis reactions of AQP4 DNA were done using 20–25 ng of target AQP4 plasmid DNA according to the manufacturer's instructions. AQP4 DNA mutations were confirmed by sequencing, and endonuclease-free plasmid DNA was purified for transfection into mammalian cells (Qiagen catalog no. 12362).

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Papers

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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