Masterpure complete dna rna purification kit

Fecal samples were directly collected from the diaper when full enteral feeding (defined as ≥150 cc/kg/day of MOM, DHM, or formula) was achieved. Samples were frozen and stored at -80°C for later analysis.
Total fecal DNA was isolated using the MasterPure Complete DNA & RNA Purification Kit (Epicentre, Madison, WI, United States) according to the manufacturer’s instructions with modifications that included a bead-beater step and enzyme incubation to increase DNA extraction as described elsewhere (Boix-Amoros et al., 2016 (link)). Total DNA concentration was measured using a Qubit^® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, United States) and normalized to 5 ng/μL for 16S rDNA gene (V3–V4 region) amplification using Nextera XT Index Kit. Amplicons were checked with a Bioanalyzer DNA 1000 chip and libraries were sequenced using a 2 × 300 bp paired-end run (MiSeq Reagent kit v3) on a MiSeq-Illumina platform (FISABIO sequencing service, Valencia, Spain). Controls during DNA extraction and PCR amplification were also included and sequenced.

Genomic DNA was isolated from a body segment or from seminal vesicles using the MasterPure complete DNA & RNA purification kit (Epicentre) and FastPrep 24 homogenizer with Lysing Matrix Y (MP Biomedicals).

Genomic DNA was purified using Masterpure™ complete DNA/RNA purification kit (Epicentre, Cambio Ltd., Cambridge) and plasmids were purified using E.Z.N.A^® plasmid mini kit (Omega Bio-tek, Inc., Norcross, GA). The DNA concentration was measured using NanoDrop™ 2000c spectrophotometer (Thermo Scientific, DE, USA). The PCR amplification was performed using Phusion^® polymerase (Thermo Fisher Scientific, Waltham, MA, USA) or Taq polymerase (Sigma, St. Louis, MO, USA) in a Arktik™ thermal cycler (Thermo Fisher Scientific, USA). Restriction digestion using XhoI and SpeI restriction enzymes and DNA ligation using T4 DNA ligase were performed as recommended by the manufactures and were obtained from New England Biolabs (Ipswich, MA, USA). The PCR products and the digested DNA fragments were separated using agarose gel electrophoresis and extracted using Montage^® gel extraction kit (Millipore, MA, USA). DNA sequencing was performed using Big Dye (Applied biosystems, CA, USA). The primers used in the PCR and sequencing reactions were synthesized by Sigma-Aldrich and are listed in Table 1.

Milk samples (5 ml) were thawed and centrifuged at 4,000 x g for 20 minutes to separate fat and cells from whey. Thereafter, total DNA was isolated from the pellets by using the MasterPure Complete DNA & RNA Purification Kit (Epicentre) according to the manufacturer’s instructions with some modifications^{57 (link)}. 250 μl of sterile saline solution and 250 μl of lysis buffer were added to the pellets, together with a mix of 150–212 μm and 425–600 μm, acid-washed glass beads (Sigma). To enhance the disruption of fungal cell walls, samples were put through three cycles of vigorous mixing in a TissueLyser II (QIAGEN) 5 min at 30 Hz, incubation in dry ice 3 minutes and 5 minutes at 65 °C in a heat block. Lysozyme (20 mg/ml) and zymolyase (0.25 mg/ml) were added to the tubes, and samples were incubated for 1 h at 37 °C. 2 μl of proteinase K were added and samples were incubated for 15 minutes at 65 °C. The reaction was stopped by putting tubes on ice and proteins were precipitated using 350 μl of MPC Protein Precipitation Solution, and discarding the pellets. DNA was precipitated using isopropanol, washed with 70% Ethanol and resuspended with 30 μl TE buffer. The total DNA isolated was quantified with a Qubit^TM 3 Fluorometer (ThermoScientific).

Total RNA, including miRNAs were isolated from sperm samples using MasterPure Complete DNA & RNA Purification Kit (Epicentre) according to the protocol recommended by the manufacturers. Total RNA was quantified using Qubit RNA HS Assay Kit (Invitrogen, USA) and measured by Qubit 2.0 fluorometer (Life Technologies, USA).

Total DNA was isolated from the sorted bacteria by using the MasterPure Complete DNA & RNA Purification Kit (Epicentre) with a previous enzymatic step with 20 mg/mL of lysozyme (Roche) and 10 U/mL of mutanolysin (Sigma) at 37 °C for 1 h, followed by glass bead beating (0.17 mm diameter) for 1 min at 4 °C. The obtained DNA was amplified with the TruePrime WGA kit (Sygnis), following the manufacturer’s instructions. Total DNA concentration was measured and normalized using a Qubit^® 2.0 Fluorometer (Life Technology, Carlsbad, CA, USA). Subsequently, the V3-V4 region of the bacterial 16S rDNA gene was amplified by PCR using Illumina adapter overhang nucleotide sequences, following Illumina protocols. After 16S rDNA gene amplification, a multiplexing step was performed with the Nextera XT Index kit (Illumina, San Diego, CA, USA). Amplicons were checked with a Bioanalyzer DNA 1000 chip, and libraries were sequenced using a 2 × 300 bp paired-end run (MiSeq Reagent kit v3) on a MiSeq-Illumina platform (FISABIO Sequencing Service, Valencia, Spain). Controls during PCR amplification were also included and sequenced.