Hotstart taq pcr mastermix

Two clinical samples harboring wild-type (YMDD, ATG coding for methionine) and mutant (YIDD, ATT coding for isoleucine) DNA sequences confirmed by direct sequencing were used for plasmids construction. Target DNA was amplified with primers (KF, L1 and L2) listed in Table 1. PCR was performed in a 25-μl reaction mixture containing 12.5 µl of 2× HotStart Taq PCR Mastermix (Tiangen, China), 0.7 µl of each primer (10 µM), 9.1 µl of ddH₂O and 2.0 µl of DNA template. Thermal cycling conditions were as follows: initial denaturation at 94°C for 3 min, then 35 cycles with denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 45 s, and a final elongation step at 72°C for 10 min. PCR products were electrophoresed on a 2.0% agarose gel and purified via PCR purification kit (Sangon, China), then cloned into pMD 18-T vector (Takara, Japan) and transformed into E. coli DH5α competent cell (Takara, Japan). The positive plasmids were sequenced and then isolated using Tiangen mini plasmid isolation kit (Tiangen, China). Plasmids were quantified by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The corresponding copy number were calculated and 10-fold serially diluted from 1×10¹⁰ copies/μl to 1×10¹ copies/μl using Easy Dilution Buffer (Takara, Japan) to generate standard concentrations.

Sanger sequencing was performed as previously reported.³⁰ First, cDNA was amplified using 2 × HotStart Taq PCR MasterMix (TIANGEN). Then the PCR products were sequenced by Sangon Biotech (Shanghai, China). Sequencing results were compared with corresponding entries in the National Centre for Biotechnology Information (NCBI) Nucleotide Database (http://www.ncbi.nlm.nih.gov/nuccore/, NFE2L2: NM_006164.5, KEAP1: NM_203500.2, CUL3: NM_003590.5). Reduplicated experiments further confirmed all of the mutations detected. Single nucleotide polymorphism (SNP) information was obtained from the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/snp/).