Sybr safe dna gel stain

Total RNA purified (2 µg) from GTC-phenol:chloroform extraction was resolved on a 12% TBE-Urea polyacrylamide gel and electrophoresed at 200 V for at least 4 h in 1× TBE buffer. The gel was post-stained with SYBR Safe DNA Gel Stain and intact RNA was confirmed using the Bio-Rad Chemi-doc. The RNA was transferred onto a nylon membrane (GE Healthcare Life Sciences) soaked in 0.5× TBE buffer via electrophoresis at 30 V for 4 h at 4°C and then crosslinked in a Strategene Auto-crosslinker with 1200 mJ UV-C. The membrane was pre-hybridized in ULTRAhyb-Oligo buffer (ThermoFisher) at 37°C for 30 min. The 35 mer DNA oligonucleotide probes were radiolabeled with ³²P-ATP at 37°C for 1 h and then purified over a G50 size exclusion column (GE Healthcare Life Sciences) following the manufacturer’s instructions. The purified oligonucleotide mix was added to the nylon membrane and hybridized at 37°C overnight. The membrane was washed four times in wash buffer (2× subsp. buffer [Sigma-Aldrich] and 0.1% SDS) at 37°C for 30 min. Afterward, the membrane was exposed on a BAS Storage Phosphor Screen and detection of the hybridized probe was performed on the LAS-3000 (Fujifilm).

The viral RNA from sera samples was extracted using the Viral Nucleic Acid Kit II (Geneaid^®, Geneaid Biotech Ltd., Taiwan) as described by the manufacturer. The extracted RNA was subjected to the nested-multiplex PCR BVD test followed by agarose gel resolution to genotype the BVDV.
The specific NS5B primers used for external reaction and nested-multiplex genotyping are listed as Table-1 [16 (link)]. The first step external reverse transcriptase reactions were performed using the AffinityScript One-Step RT-PCR Kit (Agilent, Santa Clara, CA, USA) as specified by the manufacturer. The thermal conditions for the external reactions were as follows: 45°C for 30 min of reverse transcription, 92°C for 1 min of initial denaturing followed by 40 cycles of 92°C for 20 s, 50°C for 20 s, 68°C for 45 s, and final elongation at 68°C for 5 min. The second stage of the multiplex PCR genotyping was done using HotStar Taq Mastermix Kit (Qiagen^®, Hilden, Germany). The reaction was heated to 95°C for 15 min, followed by 35 cycles of 94°C for 40 s, 56°C for 40 s, 72°C for 40 s, with a final elongation step of 72°C for 7 min.
BVDV-1 strain Singer was used as a positive control for genotype-1 in this study. All the PCR products were separated on a 1.5% agarose gel, stained with SYBR^® Safe DNA gel stain, and visualized using Gel Documentation Systems from Bio-Rad.

Iron(III) acetylacetonate (99.9%), 1,2-hexadecanediol (90%), oleic acid (>99%), oleylamine (>70%), and diphenyl ether (>99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. α-Cyclodextrin (α-CD) was supplied by Tokyo Chemical Industry (Tokyo, Japan). Branched oligoethyleneimine with molecular size of 600 Dalton (OEI-600), branched polyethyleneimine with molecular size of 25 kDa (PEI-25k), 1,1′-carbonyldiimidazole (CDI), dimethyl sulfoxide (DMSO), dimethyl ether (Et₂O), and tetrahydrofuran (THF) were supplied by Sigma-Aldrich. Deuterium oxide (D₂O), which was used as a solvent in NMR experiments, was supplied by Sigma-Aldrich.
Plasmid DNA pRL-CMV containing a Renilla luciferase reporter gene, supplied by Promega (Madison, WI, USA), was amplified in E. Coli and purified with a commercial kit (Endofree Plasmid Maxi Kit, Qiagen, Hilden, Germany). Luciferase kit was supplied by Promega. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), streptomycin, and penicillin were supplied by Sigma-Aldrich. The nucleic acid sample loading buffer and SYBR safe DNA gel stain were from Bio-Rad Laboratories (Hercules, CA, USA) and Invitrogen (Waltham, MA, USA), respectively.

Fragments of the TaGAPCp2P/3P were cloned into plasmid pAbAi (TaKaRa, Japan) to screen the cDNA library, and screening of the cDNA library was performed according to the manufacturer’s instructions (Matchmaker One-Hybrid system; Clontech Laboratories Inc., Palo Alto, CA, USA) in the presence of 20 mM Aureobasidin A (AbA).
A total of 100 ng of a 30 bp double stranded probe and 1 μg of purified TaMYB were used in the EMSA reactions. After incubation at room temperature for 40 min, the samples were loaded onto a 6% native polyacrylamide gel. Then, the gel was poststained with Invitrogen™ SYBR™ Safe DNA Gel Stain and imaged with a Bio-Rad gel documentation system to detect DNA.