Ultraclean tissue cells dna isolation kit

Genomic DNA was extracted from EDTA blood samples from calf 1, calf 2 and calf 3, as well as from the two obligate carrier dams of calf 2 and calf 3 using the UltraClean® Tissue & Cells DNA Isolation Kit (Mo Bio Laboratories Inc.) following the manufacturer’s protocol. Genomic DNA was isolated from hair roots from 403 Angus/Angus-cross animals collected in 2016 and 2017 from the original herd using a standard hair digest protocol [38 (link)].
RNA was extracted from cultured fibroblast cells from calf 2 and calf 3 and one unrelated Angus animal using the RNeasy Mini Kit (QIAGEN). Extraction was performed according to the manufacturer’s protocol with the addition of 600 μL of RLT buffer to lyse cells.
DNA and RNA concentration and purity were measured using the NanoDrop 8000 spectrophotometer (ThermoFisher Scientific).

Total DNA was extracted from the kidneys of mice A, C, D, E, and F with commercially available kits (UltraClean^® Tissue & Cells DNA Isolation kit (MO BIO Laboratories, Inc, USA); DNEasy Tissue and Blood (Qiagen) according to the manufacturers’ instructions. For total DNA extracts from mouse A, a PCR reaction mixture contained the following components with final concentration of 20 mmol/L Tris-HCl (pH 8.5 at 25 °C), 50 mmol/L KCl, 2 mmol/L MgCl₂, 200 µmol/L each dNTP (dATP, dCTP, dTTP and dGTP), 0.5 U of Taq polymerase (Fisher Biotec, Australia) and 0.2 µmol/L each primers specific for murine polyomavirus VP2 protein-coding region [7 (link)] in a final PCR reaction volume of 25 µL. The PCR was run on a Peltier thermal cycler (MJ Research, United States) with the following temperature program: an initial denaturing step at 94 °C for 5 min, then 40 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s. The PCR products were electrophoretically separated in 2% agarose E-gels (Invitrogen, Australia) and visualized with UV light [6 (link)].
A broad-spectrum papillomavirus PCR was performed on DNA extracts from mice C, D, E, and F, according to the method described in Forslund et al. (1999) [8 (link)]. Multiply primed rolling circle amplification was also performed on these same samples, following the method of Rector and co-authors (2004, 2005) [3 (link),9 (link)].

DNA extraction was performed using Ultra Clean Tissue & Cells DNA Isolation Kit according to manufacturer's instruction (Mo Bio Laboratories, Carlsbad, CA). Methylation status of CpG islands of the MMP2 promoter was assessed by MethylScreen technology using the Epitect methyl II PCR assay (Qiagen) according to manufacturer's instruction. Percentages of unmethylated and hypermethylated CpG values were calculated using a quantitation algorithm provided by the manufacturer (Epitect methyl II PCR assay Handbook - Qiagen).