Midori green advance

Manufactured by Nippon Genetics

Sourced in Germany, Japan, France

Midori Green Advance is a DNA staining solution for use in gel electrophoresis and fluorescence-based applications. It provides sensitive and specific detection of DNA fragments.

Automatically generated - may contain errors

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46 protocols using midori green advance

Genotyping Llama CSN1S1 Gene SNP

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The entire panel of 128 animals was genotyped for the SNP c.366A>G using the PCR-RFLP method. A DNA fragment 153 bp long (estimated according to EMBL Acc. N. KN269544.1) spanning from bp 2 of exon 11 to the 36th nucleotide of exon 12 of the llama CSN1S1 gene was amplified by using the primers (forward) 5′-AAGTTGTTTCCAGTACCAC-3′ and (reverse) 5′-TTGGGAGGGCATATCTT-3′, as well as the same PCR mixture conditions reported by Pauciullo and Erhardt (2015) (link). Thermal conditions were 95°C for 4 min, 35 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 20 s, with the final extension at 72°C for 5 min. Product specificity was confirmed by Midori Green Advance (Nippon Genetics, Düren, Germany) stained by 1.5% agarose gel electrophoresis.
Ten microliters of each PCR amplification was digested with 5 U of MaeII endonuclease (5′-A↓CGT-3′; New England Biolabs, Ipswich, MA) overnight at 65°C. The digested products were analyzed by electrophoresis in 3.5% agarose gel in 0.5× Tris-borate-EDTA buffer and stained with Midori Green Advance (Nippon Genetics).

Pauciullo A., Gauly M., Cosenza G., Wagner H, & Erhardt G. (2017). Lama glama α_S1-casein: Identification of new polymorphisms in the CSN1S1 gene. Journal of dairy science, 100(2).

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Phage DNA Restriction Mapping

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DNA of three phages (ACF1, ACF8, and ACF12) was digested with BamHI, EcoRI, and BsmI restriction enzymes (Fermentas, Lithuania) as recommended by manufacturer. Digestion reaction contained 500 ng DNA, 1.5 μl buffer, 2 μl restriction enzyme and up to 15 μl nuclease-free water. The mixtures were incubated at 37°C for 16 h. DNA fragments were separated by agarose (1%) gel electrophoresis in Tris-acetate-EDTA buffer, stained with Midori Green (MIDORI Green Advance, NIPPON Genetics EUROPE) 2% (v/v) and visualized by a digital imaging camera (Vilber Lourmat, France).
The in silico restriction fragment length polymorphism (RFLP) analysis was performed by pDRAW32 software (AcaClone Software)¹.

Gašić K., Obradović M., Kuzmanović N., Zlatković N., Ivanović M., Ristić D, & Obradović A. (2022). Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli. Frontiers in Microbiology, 12, 803789.

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Removal of DNA Contamination in RNA Samples

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To remove any DNA contamination, 5 µl (1 U µl⁻¹) DNAase I and 5 µl DNAse I buffer were added to each 50 µl RNA sample followed by incubation at 37 °C for 30 min. DNAse I was then inactivated by adding 5 µl EDTA (50 mM) and incubation at 65 °C for 10 min. Eventually, the RNA samples were checked for DNA contamination by PCR analysis using actin gene specific primers: 5′-AGTACGTACGTGTTGGCCATG-3′ (actin forward primer) and 5′-AGTACGTACGTGTTGGCCATG-3′ (actin reverse primer). For a PCR, 2 µl of RNA sample were mixed with 1 µl 10 µM of each forward and reverse primer, respectively, 5 µl 10× reaction buffer (Axon), 4 µl 25 mM MgCl₂, 1 µl of a mixture of 10 mM (each) dNTP, 0.5 µl (5 U µl⁻¹) Taq polymerase (Axon) and 35.5 µl ddH₂O to a final volume of 50 µl. PCR reactions were initiated by DNA denaturation for at 94 °C for 1 min, followed by 35 cycles of denaturation at 94 °C for 1 min, primer annealing at 50 °C for 45 s and elongation at 72 °C for 1 min. Final elongation was performed at 72 °C for 5 min. Genomic DNA of C. pomonella was used for positive- and water for negative-control reactions. PCR fragments were separated by gel electrophoresis and staining of DNA by Midori Green Advance (Nippon Genetics Europe GmbH, Düren, Germany).

Wennmann J.T., Pietruska D, & Jehle J.A. (2021). Transcriptome of Cydia pomonella granulovirus in susceptible and type I resistant codling moth larvae. The Journal of General Virology, 102(3), 001566.

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ERIC PCR for Bacterial Strain Identification

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ERIC PCR (Enterobacterial Repetitive Intergenic Consensus PCR) was conducted according to the protocol published by Anjay et al. (2015 (link)). The method uses long primers that are complementary to the conservative fragments of the bacterial genome which are differently distributed depending on the strain. The PCR reaction was standardized with 2.5 µl of 10x Shark buffer (DNA Gdańsk), 2.5 µl of MgCl₂ (25mM) (DNA Gdańsk), 3 µl of dNTP (2 mM) (nzytech, Portugal), 30 pmol of each primer ERIC1R and ERIC2, 1U of Hypernova DNA polymerase (Blirt), 3 µl of template DNA, and nuclease‐free water up to a final volume of 25 µl. The amplification cycles included initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 60 s, annealing at 40°C for 90 s and extension at 72°C for 60 s with a single cycle for final extension at 72°C for 7 min. Primer sequences are summarized in Table S4.
Ten microlitres of PCR products were electrophoresed on a 1.5% agarose gel in the presence of Midori Green Advance (Nippon Genetics), at 120 V for 120 min. The results were read using the Quantum ST5 Gel Documentation System (Vilber).

Kaczorek‐Łukowska E., Sowińska P., Franaszek A., Dziewulska D., Małaczewska J, & Stenzel T. (2020). Can domestic pigeon be a potential carrier of zoonotic Salmonella?. Transboundary and Emerging Diseases, 68(4), 2321-2333.

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Antimicrobial Resistance Gene Detection

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All PCR reactions were carried out with a HotStarTaq Plus Master Mix Kit (Qiagen) according to the manufacturer's protocols in a nexus gradient thermocycler (Eppendorf). The following genes were selected for the antimicrobial resistance tests: sul1, sul2, sul3, tet(A), tet(B), tet(G), bla_TEM‐1, floR, cat1, cat2, aac6, strA, and strB. In case of virulence, the investigated genes were: lpf, sivH, invA, agaF, and avaR. The list of investigated genes, primer sequences, product size, and annealing temperature are summarized in Table S2. All primers were synthesized by Genomed S.A. (Poland). Ten microlitres of PCR products were electrophoresed on a 2% agarose gel in the presence of Midori Green Advance (Nippon Genetics, Germany), at 120 V for 60 min. The results were read using the Quantum ST5 Gel Documentation System (Vilber). To confirm the specificity of the amplicons obtained, some PCR products of interest were randomly selected and purified using a CleanUp kit (A&A Biotechnology) for sequencing (Genomed).

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Genetic Sex Determination Method

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Sex was determined genetically following the method described previously (Mac e and Crouau-Roy, 2008) (link). Briefly, we extracted DNA from the samples using a XYZ kit (according to manufacturer's instructions). The reaction mix contained 0.5 mM of each primer (Primers SC1: 5 0 -CAAGCATGCATTTCAATTCCC and SC2: 5 0 -CTGCATGGGGAACATCGGAG), 2 ml of DNA, and 10 ml of HotStarTaq Master Mix (Qiagen) bringing the total volume to 20 ml. PCR was achieved through the following steps: 1) initial activation at 95 C for 5 min; 2) denaturing through 45 cycles (95 C for 1 min); 3) annealing at 55 C for 45 s; 4) elongation at 72 C for 1 min; 5) final elongation at 72 C for 5 min. PCR products were run on 1% agarose gel stained with Midori Green Advance (Nippon Genetics).

Remili A., Gallego P., Pinzone M., Castro C., Jauniaux T., Garigliany M.M., Malarvannan G., Covaci A, & Das K. (2020). Humpback whales (Megaptera novaeangliae) breeding off Mozambique and Ecuador show geographic variation of persistent organic pollutants and isotopic niches. Environmental pollution (Barking, Essex : 1987), 267.

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Pneumococcal Capsular Typing by PCR

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PCR was performed in 25-μL reaction mixtures, containing 1 U Ex Taq DNA polymerase (TaKaRa Bio, Tokyo, Japan), 0.2 mM of each deoxyribonucleoside triphosphate, 10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 0.5 μM of each primer, and 2 μL of template DNA, on a SimpliAmp^TM Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The program comprised 35 cycles of denaturation at 94°C for 30 s, primer annealing at 54°C for 90 s, and extension at 72°C for 60 s, with a final incubation at 72°C for 10 minutes [27 (link)–29 (link)]. Previously reported PCR primers were used for detecting capsular types 2 and 8 [28 (link)], 9N [29 (link)], 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F [27 (link)] (S1 Table). The PCR products were electrophoresed on agarose gels and visualized with Midori Green Advance (NIPPON Genetics, Tokyo, Japan).

Lee J., Yoon Y., Kim E.J., Lee D., Baek Y., Takano C., Chang B., Iijima T., Kilgore P.E., Hayakawa S., Hoshino T., Kim D.W, & Seki M. (2021). 23-valent polysaccharide vaccine (PPSV23)-targeted serotype-specific identification of Streptococcus pneumoniae using the loop-mediated isothermal amplification (LAMP) method. PLoS ONE, 16(2), e0246699.

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Quantitative RT-PCR Analysis of Slc5a2 and Gapdh

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After perfusion with PBS, choroid plexus and kidney tissues were isolated from mice (n = 3), and total RNA was extracted using a ReliaPrep RNA Tissue Miniprep System (Promega, Fitchburg, WI, USA). The cDNA was synthesized with reverse transcriptase using a ReverTra Ace qPCR RT Master Mix (Toyobo). Ten nanograms of cDNA were used as a template, and the specific portions of the two transcripts were amplified with GoTaq G2 Hot Start Polymerase (Promega) and primer sets specific for the SGLT2/SLC5A2 gene (Slc5a2) and the glyceroaldehyde‐3‐phosphate dehydrogenase (GAPDH) gene (Gapdh) (Table 2). PCR amplification cycle conditions were as follows: (i) 2 min at 95C; (ii) 25, 30, or 35 cycles of 30 s at 95C, 15 s at 55C, and 30 s at 72C; and (iii) 5 min at 72°C. The amplicons were electrophoresed on a 2% agarose gel, stained with Midori Green Advance (Nippon Genetics, Tokyo, Japan), and visualized with a blue LED (470 nm) transilluminator (AMZ System Science, Osaka, Japan). Amplified fragments were subjected to direct sequencing (Eurofins Genomics, Tokyo, Japan).

Chiba Y., Sugiyama Y., Nishi N., Nonaka W., Murakami R, & Ueno M. (2020). Sodium/glucose cotransporter 2 is expressed in choroid plexus epithelial cells and ependymal cells in human and mouse brains. Neuropathology, 40(5), 482-491.

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Detection of β-Lactamase Genes by PCR

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The PCR primers and conditions for the β-lactamases (Supplementary Table 3) were described previously (Senda et al., 1996 (link); Monteiro et al., 2012 (link)). PCR assays were performed in a 25 μL reaction mixture containing 0.2 mM each deoxynucleoside triphosphate, 10 mM Tris-HCl (pH 8.0), 10 mM KCl, 2.5 mM MgCl₂, 0.4 μM each primer, 2.5 U of Ex Taq DNA Polymerase (Takara, Shiga, Japan), and template DNA (2 μL). Reactions were performed on a SimpliAmp™ Thermal Cycler (Applied Biosystems). The PCR program was 5 min at 94°C; followed by 35 cycles of denaturation at 94°C for 90 s, annealing at 55°C for 30 s, and extension at 72°C; and a final extension at 72°C for 15 min, followed by storage at 4°C. The PCR products were subjected to agarose gel electrophoresis and visualized with Midori Green Advance (NIPPON Genetics, Tokyo, Japan).

Kim E.J., Lee J., Yoon Y., Lee D., Baek Y., Takano C., Sakai J., Iijima T., Kanamori D., Gardner H., McLaughlin R.E., Kilgore P.E., Nakamura A., Ogihara T., Hayakawa S., Hoshino T., Kim D.W, & Seki M. (2023). Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria. Frontiers in Cellular and Infection Microbiology, 12, 1000445.

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Gel Retardation Assay for Nucleic Acid Binding

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To test the binding efficiency of nucleic acids to CS, a gel retardation assay was carried out. Briefly, nanocomplexes were prepared at different P/N charge ratios as described above and loaded onto a 1.5% agarose gel in 0.5× TBE buffer supplemented with 0.006% Midori Green Advance (Nippon Genetics Europe GmbH, Dueren, Germany) and electrophoresed at 128 V for 40 min. Finally, the nucleic acid bands were visualized in a BioDocAnalyze System (Analytik Jena, Jena, Germany).

Kolonko A.K., Bangel-Ruland N., Goycoolea F.M, & Weber W.M. (2020). Chitosan Nanocomplexes for the Delivery of ENaC Antisense Oligonucleotides to Airway Epithelial Cells. Biomolecules, 10(4), 553.

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