Innuprep dna mini kit

Genomic DNA of a hABCB1 mouse was purified using an innuPREP DNA Mini Kit (AnalytikJena AG, Germany). The humanized genomic locus was amplified in 25 µL reactions using TaKaRa LA TaqDNA Polymerase (TaKaRa Bio USA Inc.) according to manufacturer recommendations using 0.4 µM of each primer and an annealing temperature of 60 °C, resulting to single-banded PCR products. After amplification, the remaining primers and dNTPs were digested. The amplified fragments were sequenced using the Sanger process utilizing a primer walking strategy. For the sequencing of exons 3 and 4, PCR reactions were run with 12.5 ng template DNA in a total volume of 12.5 µL. PCR reactions including 0.3 µM of each PCR primer, 200 µM dNTPs, 1.5 mM MgCl2 and 0.02 U/µL KAPA2G Robust polymerase. 35 cycles were run for each PCR with an initial denaturation step of 180 s at 95 °C followed by 35 cycles with a denaturation step for 20 s at 95 °C, an annealing step at 61 °C for 20 s, an elongation step for 100 s at 72 °C and a final elongation step at 72 ° for 45 s. Excess dNTPs and primers were removed from PCR reactions and Sanger sequencing was performed. All primers used are listed in Supplementary Table 1. Amplification, primer design, and sequencing were realized by Microsynth AG (Switzerland).