Applied biosystems proflex pcr system

cDNA was synthesized from the individual chestnut teal samples using 5 µL of nucleic acids (obtained after virus enrichment, as described above) as the starting material, using 10× PathAm FluA RT Enzyme Mix at 45 °C for 60 min, and then at 95 °C for 1 min, as described in the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). Synthesized cDNA was then amplified using 25× PathAmp FluA PCR Primer Mix and 5 U/μL SuperTaq plus PCR Enzyme (Applied Biosystems) at 95 °C for 4 min, with 40 cycles of 95 °C for 15 s, 55 °C for 30 s, and 68 °C for 2 min, with a final 68 °C step for 7 min using an Applied Biosystems ProFlex PCR system (Thermofisher Scientific, Waltham, MA, USA). The amplified products were purified using 1.5× Agencourt AMPure XP kit (Beckman Coulter, Brea, CA, USA), and 5 µL of the purified products were then fragmented and amplified using the same SeqPlex Kit (Sigma), as described above, following the manufacturer’s protocol [36 (link),37 (link)].

One hundred msDNA loci were randomly selected, and PCR was performed using an Applied Biosystems™ ProFlex™ PCR System (Thermo Fisher Scientific, Foster City, CA, USA). PCR was performed using 20 ng gDNA; 0.5 units AccuPower^® PCR PreMix (Bioneer, Daejeon, Republic of Korea); 0.5 μM forward primer labeled fluorescent dyes FAM, HEX, TAMRA, and ATTO565; and 0.5 μM reverse primer. PCR condition was as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 20 s, annealing at 55 °C for 20 s, and extension at 72 °C for 20 s. After 35 repetitions, the final extension was performed at 72 °C for 10 min, and the temperature was held at 8 °C. Each amplified PCR product was electrophoresed on 2% agarose gel to confirm the presence or absence and size of the amplified fragment. The PCR fragments were prepared by mixing a GeneScan™ 500 ROX size standard ladder (Thermo Fisher Scientific) and HiDi™ formamide and performing denaturation at 95 °C for 2 min, followed by termination at 4 °C. The allele sizes were determined using an Applied Biosystems™ ABI 3730xl DNA Analyzer (Thermo Fisher Scientific). Genotyping was performed using GeneMapper ver. 5 software [31 ].

For NGS, the samples were processed, and virus particles enriched using a protocol optimised in our laboratory described previously³⁸ . Briefly, the samples were homogenised at 25 Hz for 2 min followed by centrifugation at 17000 g for 3 min and filtered using a 0.8 um Polyethersulfone (PES) Spin-column filter. The sample was then divided into two, one being ultracentrifuged at 178,000 g for 1 h and the other not ultracentrifuged. Both were then treated with benzonase and micrococcal nuclease for 2 h to enrich for nucleic acids protected in virus particles³⁸ . Nucleic acids (both DNA and RNA) were extracted using QIAamp Viral RNA Mini Kit (Qiagen). An initial RNA denaturation step of 95 °C for 3 min followed by snap-cooling in an ethanol-ice bath (minus 20 °C) was carried out before cDNA synthesis and DNA amplification using the SeqPlex RNA Amplification Kit (Sigma) as per the protocol described³⁸ . For PCR, cDNA synthesis of nucleic acids extracted from the individual faecal samples were conducted by using SuperScript™ VILO™ Master Mix (Invitrogen) at 25 °C for 10 min, 42 °C for 60 min, 85 °C for 5 min and hold at 4 °C in Applied Biosystems ProFlex PCR system (Thermofisher Scientific).

Genomic DNA (gDNA) from both wild and engineered B16F10 cells was isolated with a gDNA extraction kit (DN1002, Aidlab, China) according to the manufacturer's instructions. The quantity and quality of DNA were measured by the NanoDrop One instrument (Thermo Scientific, USA). The DNA samples were amplified by PCR using AmpliTaq Gold Fast PCR Master Mix (Thermo Fisher Scientific, USA) on an Applied Biosystems ProFlex PCR system (Thermo Fisher Scientific, USA). The primers were designed according to the website https://www.ncbi.nlm.nih.gov/tools/primer‐blast/, and the sequences are listed in Table S1, Supporting Information. The length of the predicted products was ≈522 bp. The PCR mixtures were then separated by 3% agarose gel electrophoresis and imaged with a UV transilluminator.

Measurements of the KIT D816V VAF on DNA, derived from peripheral blood (PB) and biopsies, were performed using the QuantStudio™ three-dimensional (3D) dPCR system in combination with the Applied Biosystems ProFlex PCR System (ThermoFisher Scientific, Waltham, MA, USA). Per sample, a 15 µL reaction volume was prepared. The volume included 7.1 µL of 10 ng/µL DNA, 7.5 µL of QuantStudio™ 3D Digital PCR Master Mix v2 (ThermoFisher Scientific, Waltham, MA, USA) and 0.4 µL of KIT D816V specific Taqman gene expression assay (ID: Hs000000039_rm, ThermoFisher Scientific Waltham, MA, USA).