QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Protocol tips
- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Publication protocol

Construction of the PAH_c.1222C>T vector and establishment of the PAH_c.1222C>T COS-7 cell line
A wild-type PAH cDNA was cloned into the pLVX-EF1α-IRES-Puro vector (Clontech) using Xbal and BamHI cleavage (Thermo Fisher Scientific) after the amplification of cDNA from the pCMV-FLAG-PAH wild-type plasmid. The PAH_c.1222C>T variant in the PAH cDNA sequence was introduced by site-directed mutagenesis using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) and confirmed by Sanger DNA sequencing (GATC Biotech). EF1α forward and IRES reverse sequencing primers (Thermo Fisher Scientific) were used to sequence the PAH_c.1222C>T variant.

COS-7 cells were cultured at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. One day prior to transfection, 2 × 106 cells were seeded and cultured in one dish of 10 cm diameter. The transfection of one dish utilized 45 μL Fugene HD reagent (Promega) and 15 μg PAH_c.1222C>T plasmid or the plasmid with the wild-type sequence as a control. Forty-eight hours post transfection, the growth medium was replaced with complete growth medium containing 10 μg/mL puromycin as the selective agent. After the non-transfected COS-7 cells had died, a single PAH_c.1222C>T COS-7 clone was isolated using limiting dilution cloning technique performed in 96-well plates with 10 μg/mL puromycin remaining in the culture medium. The clone was characterized through RT-PCR, and expanded in 75 cm2 cell culture flasks.

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