Nano drop nd 1000 spectrophotometer v3

Total RNA was extracted from MV4-11, MV4-11R-cep, and MV4-11R-cep + 5-Aza cells using the Rneasy® Mini Kit (Qiagen, Valencia, California, USA), the purity and concentration was measured with a NanoDrop ND-1000 spectrophotometer V3.3.0 (NanoDrop Technologies, Berlin, Germany).

One gram of colon content was collected and immediately frozen at −80 °C until processed. Bacterial DNA was extracted from frozen samples using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) and following the manufacturer’s instructions. Fecal samples were mixed with 1 mL InhibitEX buffer in SK38 tubes and processed by using the Precellys^® 24 homogenizer for 2 × 30 s at 6500 rpm and 10 s delay between cycles. Lysis was completed at 95 °C for 5 min. Finally, DNA was eluted in 40 μL elution ATE buffer. Once DNA was extracted, DNA concentrations were measured with Qubit^® 4.0 fluorometer (Invitrogen) and dsDNA HS (high sensitivity) Assay Kit (Invitrogen). DNA purity was assessed by measuring the A260/A280 with NanoDrop^® ND-1000 Spectrophotometer V3.0.1 (Thermo Scientific, Waltham, MA, USA) and monitored on 1% agarose gels.

Bacterial DNA was extracted from faecal samples using the NZY Soil gDNA Isolation kit (NZYTech, Lisboa, Portugal) following the manufacturer’s instructions and minor modifications. Stool samples (120–180 mg) were mixed with 700 µL NSL1 buffer in NZYSpin Soil Bead Tubes and processed by using the Precellys^® 24 homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) for 2 × 30 s at 6500 rpm and 10 s delay between cycles. Once DNA was extracted, the concentration of the DNA was measured with a Qubit^® 4.0 fluorometer (Invitrogen, Thermo Fisher Scientific, MA, USA). DNA purity was assessed by measuring the A260/A280 with a NanoDrop^® ND-1000 Spectrophotometer V3.0.1 (Thermo Scientific, MA, USA) and monitored on 1% agarose gels.

DNA was extracted from 180 mg fecal samples using a QIAamp Fast DNA Stool Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Genomic DNA was cleaned and concentrated by a Genomic DNA Clean & Concentrator Kit-10 (Zymo Research, Irvine, CA, USA), according to the manufacturer’s directions. DNA concentration and purity were determined using a NanoDrop ND-1000 Spectrophotometer V3.7 (Thermo Fisher Scientific, Waltham, MA, USA) and the integrity of total DNA samples was evaluated with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). DNA samples were stored at − 20 °C until further analysis.

Genomic DNA was isolated using the traditional Cetyltrimethylammonium bromide (CTAB) method as previously described (Somerville et al., 2005 (link)). Extracted DNA was quantified using Qubit 3.0 fluorometer (Life Technologies, USA) according to manufacturer’s instructions and DNA purity was determined on a NanoDrop ND-1000 Spectrophotometer V3.7 (Thermo Scientific, Wilmington U.S.A) following manufacturer’s instructions. DNA purity was checked at absorbance 260 nm and 280 nm by calculating a ratio of A260/A280. DNA quality was analyzed on 1.5% Agarose Gel electrophoresis and visualized under UV light following ethidium bromide staining.

For qRT-PCR, samples were collected daily until the moisture content dropped to approximately 9–10% for the soil grown plants Tienshan and Kopu and 32% for both the PS and NPS treatments of vermiculite:perlite grown Huia. Total RNA was extracted from different samples using the Hot Borate method (Hunter and Reid, 2001 (link); Moser et al., 2004 (link)). The RNA/DNA concentration was determined by measuring the absorbance at 260 nm (A₂₆₀) using a NanoDrop ND-1000 spectrophotometer V3.6 (Thermo Scientific, United States). Genomic DNA-free RNA samples were prepared according to the manufacturer’s instructions using an RNase-free recombinant DNase treatment (Roche Applied Sciences, Roche Diagnostics GmbH, Mannheim, Germany). Total RNA (2–10 μg) was mixed with 5 μL of 10× incubation buffer and 1 μL of DNase I (10 U), 1 μL of Protector RNase Inhibitor (10 U) and water to a final volume of 48.4 μL and incubated at 37°C for 20 min. The reaction was stopped by the addition of 1.6 μL of 0.25 M EDTA pH 8.0 (to make a final concentration of 8 mM) and was heated at 75°C for 10 min.