Mag bind blood tissue dna hdq kit

We obtained 354 tissue samples collected by the California Department of Fish and Wildlife between 2011–2017 from pumas either hit by car (~6%), found dead (~2%), poached (<1%), or through depredation permits (>90%), which had never been used in any previous genetic survey. Samples were well‐distributed throughout the state, except for smaller populations in smaller mountain ranges. To bolster our sample size in the Los Angeles region of southern California, we added the only remaining DNA extracts (N = 144) from pumas collected between 2002–2015 (Riley et al., 2014 (link); Vickers et al., 2015 (link)). After genomic and bioinformatic filtering (described below), we retained 401 out of 498 samples in the final dataset, which spanned the majority of puma habitat in California, excluding desert regions (Figure 1). For samples that lacked a precise GPS location, we used the nearest address or town where they were collected as their GPS point. Samples were stored at −80°C until DNA was extracted using Omega Bio‐tek Mag‐Bind Blood & Tissue DNA HDQ Kits (Omega Bio‐tek, #M6399‐01), with a manufacturer‐designed protocol for the Kingfisher Duo Prime (ThermoFisher Scientific, #5400110) automated DNA purification system. We measured the concentration of DNA from each sample using a Qubit 3.0 fluorometer (Invitrogen, #Q33216) with Qubit dsDNA high‐sensitivity kits (Invitrogen, #Q32854).

Genomic DNA was extracted from both blood and saliva samples using Mag-Bind® paramagnetic particles (Mag-Bind Blood & Tissue DNA HDQ kit, Omega Biotek, GA) on a Biomek® NX^P liquid handler (Beckman Coulter). In brief, 400 μL of each saliva sample (diluted with OraCollect bacteriostatic solution) or 200 μL of each EDTA-containing whole blood sample was processed for the extraction according to the instructions provided in the kit. Extracted DNA samples from both specimen types were eluted in 50 μL of elution buffer and were quantified by measuring their UV absorbance at wavelength 260 nm on a spectrophotometer (SpectraMax Plus). DNA samples were also quantified on a Qubit® 3.0 fluorometer (Thermo Fisher Scientific, CA) using the Qubit dsDNA HS quantification kit (Thermo Fisher), according to the manufacturer's instructions. The purity and quality of the DNA samples were assessed by reading each sample's OD at 260 nm and 280 nm on the spectrophotometer. The OD₂₆₀/OD₂₈₀ ratio for each DNA sample was calculated, and a ratio above 1.7 was considered sufficient purity [10 (link)]. After extraction, all the DNA samples were stored at −20°C.

Genomic DNA (gDNA) was isolated using the KingFisher Flex Purification System with the Mag-Bind® Blood & Tissue DNA HDQ Kit (Omega Bio-Tek). The gDNA concentrations were quantitated by Qubit.
For PCR amplification, gDNA was divided into 100 μL reactions such that each well had at most 10 μg of gDNA. Plasmid DNA (pDNA) was also included at a maximum of 100 pg per well. Per 96-well plate, a master mix consisted of 150 μL DNA Polymerase (Titanium Taq; Takara), 1 mL of 10x buffer, 800 μL of dNTPs (Takara), 50 μL of P5 stagger primer mix (stock at 100 μM concentration), 500 μL of DMSO (if used), and water to bring the final volume to 4 mL. Each well consisted of 50 μL gDNA plus water, 40 μL PCR master mix, and 10 μL of a uniquely barcoded P7 primer (stock at 5 μM concentration). PCR cycling conditions were as follows: (1) 95 °C for 1 min; (2) 94 °C for 30 s; (3) 52.5 °C for 30 s; (4) 72 °C for 30 s; (5) go to (2), ×27; (6) 72 °C for 10 min. PCR primers were synthesized at Integrated DNA Technologies (IDT). PCR products were purified with Agencourt AMPure XP SPRI beads according to manufacturer’s instructions (Beckman Coulter, A63880), using a 1:1 ratio of beads to PCR product. Samples were sequenced on a HiSeq2500 HighOutput (Illumina) with a 5% spike-in of PhiX.