Phusion Site-Directed Mutagenesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Phusion Hot Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures due to higher salt concentrations in its buffer. Hence follow manufacturer instruction strictly.

Upstream tips
Phusion Hot Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures due to higher salt concentrations in its buffer. Hence follow manufacturer instruction strictly.

Publication protocol

Constructs and transfection
The full length human ALK cDNA was purchased from ATCC and subcloned into the mammalian expression vector pcDNA3.1. Point mutations F1174L and R1275Q were introduced by site-directed mutagenesis, using the Phusion site-directed mutagenesis kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the following oligonucletides: P-TCATCAGCAAATTAAACCACCAGA and P-TCAGGGCTTCCATGAGGAAATC to generate F1174L substitution; P-GGATGGCCCAAGACATCTACA and P-CGAAGTCTCCAATCTTGGCCA to introduce R1275Q mutation. Transfection was then performed using Lipofectamine 2000 reagent (Invitrogen), according to manufacturer’s instructions.

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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Phusion Site-Directed Mutagenesis Kit below.

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Videos

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