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 of the C1 Domain and the Full-length RasGRP2
Point mutations of the amino acid residues were introduced using the GeneTailor® and GeneArt® site-directed mutagenesis system (Invitrogen) according to the manufacturer's instructions. To generate the C1 domain and full-length mutants of RasGRP2, the above mentioned wild-type C1 and full-length constructs in pEGFP-C3 and pGEX-2T were used. Single mutations (S8Y) were introduced in one step, and double (S8Y/A19G), triple (N7T/S8Y/A19G and S8Y/A19G/I21L), and quadruple mutants (N7T/S8Y/A19G/I21L) were generated in a stepwise fashion using single and double mutants as templates. The presence of mutations was verified by DNA sequencing (DNA Minicore).

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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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