Primestar max dna polymerase master mix

Human full-length and Δ3-22 CYP3A4, as well as the R212A and T224A variants were produced as reported previously (21 (link), 39 (link)). The F219A mutation was introduced using PrimeSTAR Max DNA Polymerase master mix (Takara Bio) and confirmed by sequencing. The F219A variant of Δ3-22 CYP3A4 was expressed and purified using the protocol developed for WT CYP3A4 (39 (link)). All proteins used in the study had R_z (A_{416/280 nm} ratio) of 1.5 or higher.

Genomic DNA was extracted from hepatocytes using the Qiagen DNeasy kit (Cat# 69504) according to the manufacturer’s protocol and quantitated with the Qubit dsDNS HS kit (Invitrogen, Cat# Q32854). A positive control plasmid was constructed for the nested PCR experiments. This included the human PAH payload, the flanking homology arms, and 500bp of human genomic sequence to the left and right of the homology arms. For a negative control, 100 ng of human genomic DNA from Clontech was used. 100ng genomic DNA from HuLiv mice was used for PCR. PrimeStar Max DNA polymerase master mix (Takara, Cat# R045B) were used for the PCR reaction. Primer sequences are listed in S1 Table.