Eva green pcr master mix

Manufactured by Bio-Rad

Sourced in United States, Germany

EVA Green PCR Master Mix is a ready-to-use solution designed for real-time PCR applications. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, to perform PCR reactions. The mix includes the EVA Green fluorescent dye, which binds to double-stranded DNA, allowing for real-time monitoring of the amplification process.

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Lab products found in correlation

Mastercycle, by Eppendorf (2 mentions) Revetra ace kits, by Toyobo (1 mentions) Isogen, by Nippon Gene (1 mentions) Applied biosystems 7500 real time qpcr system, by Thermo Fisher Scientific (1 mentions) Cfx96 connect, by Bio-Rad (1 mentions) Wizard sv genomic dna purification system, by Promega (1 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions) Rotor gene 6000, by Qiagen (1 mentions)

7 protocols using eva green pcr master mix

Real-Time PCR Expression Analysis

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Real-time PCR was carried out using an Eppendorf Master Cycle (Hamburg, Germany) instrument and EVA Green PCR Master Mix (Bio-Rad, Milan, Italy) according to the manufacturer’s instructions. At the end of the amplification process, a melting stage was added. There was no non-specific amplification as determined by the melting curve. All samples were tested in triplicate and β-actin was used as reference genes for data normalization for TNF-α and IL-6. The quantity for each target mRNA was calculated by the ΔCt method normalized on the basis of the β-actin and RPL30. The primer sequences were as follows:

TNF-α for: 5′-GGTGCTTGTTCCTCAGCCTC-3′

rev: 5′-AGATGATCTGACCTGCCTGGG-3′

IL-6 for: 5′-ATTCTGCGCAGCTTTAAGGA-3′

rev: 5′-AACAACAATCTGAGGTCGCC-3′

β-actin for: 5′-GGACTTCGAGCAAGAGATGG-3′

rev: 5′-GATGGAGTTGAAGGTAGTTTCG-3′

Attia H., Finocchi F., Orciani M., Mehdi M., Zidi Jrah I., Lazzarini R., Balercia G, & Mattioli Belmonte M. (2021). Pro-inflammatory cytokines and microRNAs in male infertility. Molecular Biology Reports, 48(8), 5935-5942.

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Gene Expression Analysis of RPESCs

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Real-time PCR was performed with a Master Cycle (Eppendorf, Hamburg, Germany) apparatus using EVA Green PCR Master Mix (Bio-Rad, Milano, Italy) according to the manufacturer's instructions. Conditions were as follows: denaturation 98 °C for 2 min and 40 cycles of 98 °C for 60 s and 60 °C for 60 s. A melting stage was added at the end of amplification. There was no non-specific amplification as determined by the melting curve. All samples were tested in triplicate with the reference genes β-actin and RPL30 for data normalization. Genes and related primer sequences (SOX2, KLF4, c-MYC, RPE65, CRALBP, PEDF, OTX2, MITF, p21 and p53) were as described previously [58 (link)]. The mRNA expression level of all tested genes was analyzed in young and senescent RPESCs with the 2^-ΔΔCt method: Δ (ΔCt) = ΔCt (senescent) − ΔCt (young). The relative expression values of the genes of interest are reported as mean ± standard deviation (SD) of three independent experiments.

Lazzarini R., Nicolai M., Pirani V., Mariotti C, & Di Primio R. (2018). Effects of senescent secretory phenotype acquisition on human retinal pigment epithelial stem cells. Aging (Albany NY), 10(11), 3173-3184.

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Quantitative Analysis of Indoleamine Expression

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Total RNA was extracted from cell lines with ISOGEN (Nippongene, Tokyo, Japan) and reverse transcription-PCR was performed using Revetra Ace Kits (Toyobo, Tokyo, Japan). Real time PCR was performed using the EvaGreen PCR Master Mix (Bio-Rad Technologies, CA, USA). The mRNA expression of target genes was normalised to GAPDH. The primer sequences were as follows: GAPDH, sense 5ʹ-TGCACCACCAACTGCT-3ʹ, antisense 5ʹ-GGCATGGACTGTGGTC-3ʹ; Ido1, sense 5ʹ-CCCACACTGAGCACGG-3ʹ, antisense 5ʹ-TTGCGGGGCAGCACCT-3ʹ; Ido2, sense 5ʹ-GCCCAGAGCTCCGTGC-3ʹ, antisense 5ʹ-TGGGAAGGCGGCATGT-3ʹ.

Yamamoto Y., Yamasuge W., Imai S., Kunisawa K., Hoshi M., Fujigaki H., Mouri A., Nabeshima T, & Saito K. (2018). Lipopolysaccharide shock reveals the immune function of indoleamine 2,3-dioxygenase 2 through the regulation of IL-6/stat3 signalling. Scientific Reports, 8, 15917.

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Quantification of IDO1 mRNA Expression

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To analyze the amount of IDO1 mRNA, real-time PCR was performed by the Applied biosystems 7500 real-time qPCR system (Applied Biosystems, Foster City, USA)using the EvaGreen PCR Master Mix (BioRad, Tokyo, Japan). The mRNA level of IDO was normalized to that for β-actin. Primers used in real-time PCR were as follows-IDO1:5′-GCATTTTTCAGTGTTCTTCGCATA-3′ (sense) and 5′-CATACACCAGACCGTCTGATAGCT-3′ (antisense); β-actin:5′-TGCACCACACCTTCTACAATGA-3′ (sense) and 5′-CAGCCTGGATAGCAACGTACAT-3′(antisense).

Yamamoto R., Yamamoto Y., Imai S., Fukutomi R., Ozawa Y., Abe M., Matuo Y, & Saito K. (2014). Effects of Various Phytochemicals on Indoleamine 2,3-Dioxygenase 1 Activity: Galanal Is a Novel, Competitive Inhibitor of the Enzyme. PLoS ONE, 9(2), e88789.

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Quantification of Stenotrophomonas maltophilia in Amoebae

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Samples were taken at different post-infection times and total DNA was extracted using Wizard SV Genomic DNA Purification System (Promega, Charbonnières-les-Bains, France). Abundance of S. maltophilia inside amoebae was quantified in duplicate using qPCR with a set of mono-copy gene-specific primers: smeD3: 5’ -CCAAGAGCCTTTCCGTCAT- 3’ and smeD5: 5’-TCTCGGACTTCAGCGTGAC-3’ [32 (link)]. qPCR amplification was performed using CFX-96 Connect (Bio-Rad, Marnes-la-Coquette, France) in a 25 μl volume containing 10 μL of Eva Green PCR Mastermix (Bio-Rad, Marnes-la-Coquette, France), 20 pmol of each primer and 5 μL of DNA template. The amplification conditions were as follows: 98°C for 15 minutes, followed by 45 cycles of 98°C for 10 seconds, 63°C for 20 seconds and 72°C for 15 seconds. Fluorescence was measured at the end of each cycle at 72°C and a melting curve analysis (65–98°C) was performed at the end of the amplification procedure.

Denet E., Vasselon V., Burdin B., Nazaret S, & Favre-Bonté S. (2018). Survival and growth of Stenotrophomonas maltophilia in free-living amoebae (FLA) and bacterial virulence properties. PLoS ONE, 13(2), e0192308.

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Gene Expression Analysis by qRT-PCR

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Total RNA was extracted from cells using TriFast reagent (30-2010, peqGOLD). cDNA was synthesized from 1 µg of total RNA using iScript cDNA synthesis kit (170-8890, Bio-Rad) following the manufacturer's instructions. Gene expression was analyzed by quantitative RT-PCR using the EVA Green PCR Master MIX (172-5201, Bio-Rad) with the Mx3000P detection system and relative quantification software (Stratagene). The expression levels of each gene were determined using the comparative Ct method and normalized to β-actin, as internal control. The primers are listed in Table 1.

Huang H., Koelle P., Fendler M., Schroettle A., Czihal M., Hoffmann U, & Kuhlencordt P.J. (2014). Niacin Reverses Migratory Macrophage Foam Cell Arrest Mediated by oxLDL In Vitro. PLoS ONE, 9(12), e114643.

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Detecting BEST1 Gene Rearrangements

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Real-time quantitative PCR (RQ-PCR) and MLPA analysis were carried out to detect any genomic DNA rearrangements of the BEST1 gene in two patients with no mutations identified and in six ARB patients with only one mutation identified. The RQ-PCR reactions were performed on a Rotor-Gene 6000 instrument (Corbett Research, Mortlake, NSW, Australia) in a 10-lL final volume, containing 300-nM primers and 1 lL (100 ng) genomic DNA, using the Eva Green PCR Master Mix (Bio-Rad, Hercules, CA, USA), as we previously described. 22 The primers information was summarized in Supplementary Table S1. Each assay was performed in triplicate. The human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene was used as an internal control. The relative quantitation (RQ) of the target gene was accomplished using RQ manager software (Bio-Rad) and was calculated with the 2 ÀDDCt method. The threshold value for normal was set at 0.8 to 1.3. The ranges of RQ values for deletions and duplications were defined as 0.45 to 0.74 and 1.6 to 1.8, respectively. MLPA assay was performed with a SALSA MLPA probemix P367-A2 BEST1-PRPH2a (Amsterdam, the Netherlands), following the manufacturer's protocols; this kit contains probes for each exon of the BEST1 gene.

Tian L., Sun T., Xu K., Zhang X., Peng X, & Li Y. (2017). Screening of BEST1 Gene in a Chinese Cohort With Best Vitelliform Macular Dystrophy or Autosomal Recessive Bestrophinopathy. Investigative ophthalmology & visual science, 58(9).

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