Quanti it pico green dsdna assay kit

MV samples (200 μL, 35 μg of protein) were treated with DNase I (Ambion™ DNase I (RNase-free) Invitrogen™) at 37°C for 20 min, and subsequently, 450 μL of TE buffer (25:10 Tris-EDTA) were added. Lysis was induced by amendment of 50 μL of lysozyme (50 mg/mL) (Thermo Scientific), 25 μL of 5 M NaCl, and incubation at 37°C for 30 min. Then, 50 μL of 20% SDS, 5 μL of proteinase K (20 mg/mL), and 1 μL of RNase A (PureLink™ RNase A, 20 mg/mL, Invitrogen™) were added. The mixture was incubated for 1 h at 37°C, and DNA was extracted by using phenol–chloroform and isopropanol precipitation. The DNA was resuspended by adding 50 μL of nuclease-free water and quantified using the Quanti-iT™ PicoGreen dsDNA Assay Kit (Molecular Probes, Life Technologies) through an ELISA plate reader (Synergy H1, Biotek).

For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.

For the extraction of genomic DNA approximately 25 mg of dried root material of each tomato accession was weighed and inserted in a 96 deep well plate. Samples were freeze dried for 1 week using a freeze dryer (Christ Alpha 1-4 LD plus, Germany). For the extraction of DNA a Nucleospin 96 Plant II kit (Macherey-Nagel, Germany) was used and the manufacturer's protocol was followed. The quality of the gDNA was checked on a 1.5% agarose gel. The concentration of the gDNA was calculated using the Quanti-iTTM PicoGREEN dsDNA assay kit (Invitrogen). gDNA was diluted in TE buffer pH 8.0 (10 mM Tris and 1 mM EDTA) and stored at 4°C.

For use as quantitative standards, the products of the avian HMBS-based qPCR on DNA extracted from the liver of a SPF AA broiler chicken were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). To confirm the avian HMBS-based qPCR an aliquot of the products was sequenced at GenScript (Nanjing Jiangsu, China) and the remainder quantified (ng/ml) with the Quanti-iT ^TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA). After using the molecular mass of the HMBS gene to calculate the molarity of the solution, dilutions were made to give solutions containing 10⁴, 10³, 10², 10¹, 10⁰ gene copies per reaction. These were amplified with the avian HMBS-based qPCR in triplicate to determine the detection limit of the PCR.

A DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used to extract and purify genomic DNA from seeds or seed powders in accordance with manufacturer's instructions. DNA purity was checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA), and sample concentrations further quantified using a Versafluor Fluorometer (Bio-Rad, Hercules, CA, USA) with a Quanti-iT^TM PicoGreen® dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA).

Genomic DNA was isolated from peripheral blood collected in EDTA tubes, using Perfect Pure DNA Blood kit (5 Prime) accordingly to the manufacturer’s recommendations and quantitated with Quanti-iT^TM PicoGreen^® dsDNA Assay kit (Invitrogen). All sample concentrations were normalized to 60 ng/μl and 1 μl of each DNA samples was analyzed using DMET plus arrays (Affymetrix Inc., Santa Clara, CA, US) according to the manufacturers’ indication as previously described by Di Martino et al. [17 ]. Briefly, the DMET Plus assay (Affymetrix Inc., Santa Clara, CA, US) uses Molecular Inversion Probe (MIP) technology to amplify the sequence-specific targets for each 1936 marker. The resulting target DNA were then enzymatic fragmented, labeled and hybridized to the arrays containing allele-specific oligonucleotides. Finally the DMET plus arrays were scanned with the GeneChip Scanner 3000 (Affymetrix Inc., Santa Clara, CA, US) to obtain the intensity data by CEL files. Genotypes were calculated by DMET Console software version 1.1 using the Dynamic Genotype Boundaries algorithm (Affymetrix Inc., Santa Clara, CA, US). Patients with a call rate less than 95% were excluded from further analysis.