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

Cloning of Human MDR3 and Site-directed Mutagenesis
We cloned human wild type MDR3 (NM_000443.3) as described previously (18). Site-directed mutagenesis was carried out with the QuikChange® XL (Agilent Technologies) and the Phusion site-directed mutagenesis kit (Thermo Scientific), respectively. To generate the ATPase-deficient mutant, we exchanged Glu-558 and Glu-1207 of the conserved Walker B motif against Gln with the primer pair as described before (18). The Q1174E mutant was introduced into MDR3 with the Phusion site-directed mutagenesis kit (Thermo Scientific) using forward primer 5′-GATAAGGGGACTGAGCTCTCAGGAGGTCAAAAAC-3′ and the reverse primer 5′-CCCACTCTTGTTTCATATTTGTGGGGTAACG-3′. The sequences of all constructs were verified by DNA sequencing (GATC Biotech).

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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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