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Hotstart it sybr green qpcr master mix

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The HotStart-IT SYBR Green qPCR Master Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) analysis. It contains all the necessary components, including a chemically modified hot-start DNA polymerase, SYBR Green I dye, and optimized buffer system, to perform sensitive and specific qPCR reactions.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (5 mentions) Lightcycler 480, by Roche (2 mentions) Hot start ex taq polymerase, by Takara Bio (2 mentions) Epitect bisulfite kit, by Qiagen (2 mentions) Purelink genomic dna isolation kit, by Thermo Fisher Scientific (2 mentions) Pcr cloning kit, by Qiagen (2 mentions) Epimark 5 hmc and 5 mc analysis kit, by New England Biolabs (2 mentions) Prism 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions) 7900 real time pcr system, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions) 24 well plate, by Corning (1 mentions) Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions) Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (1 mentions) Iq5 real time pcr system, by Bio-Rad (1 mentions) E z n a fungal dna mini kit, by Omega Bio-Tek (1 mentions) Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions) Abi 7300 system, by Thermo Fisher Scientific (1 mentions) Nucleospin rna 2 kit, by Macherey-Nagel (1 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

SYBR Green (3216 citations) SYBR Green mix (375 citations) SYBR Master Mix (135 citations) QPCR Master Mix (38 citations) SYBR qPCR Mix (26 citations) QPCR SYBR-Green (2 citations)

12 protocols using hotstart it sybr green qpcr master mix

1

Phenylalanine tRNA Gene Assay for Bacterial Identification

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Primers for detecting phenylalanine transfer RNA (tRNAphe) gene sequences of S.ii 25124, L. plantarum A6, and W. confusa 17 were designed by Applied Biosystems.
Real-time PCR assays were performed on an Applied Biosystems Prism 7500 Real-Time PCR System (Applied BiosystemsTM, Foster City, CA, USA). qPCR was realized using HotStart-IT SYBR Green qPCR Master Mix (Thermo Scientific, Carlsbad, CA, USA). The 25 µL reaction volume consisted of 12.5 μL of 2X HotStart-IT SYBR Green qPCR Master Mix, 0.5 μM of forward and reverse primers, and 100 to 0.01 ng of DNA were mixed. PCR conditions were denaturation at 95 °C for 10 min, followed by 40 cycles of amplification: 15 s at 95 °C, 1 min at 60 °C, and 72 °C for 1 min. Fluorescence was detected at the end of the elongation phase for each cycle.
Bolaños-Núñez S., Santiago-Urbina J.A., Guyot J.P., Díaz-Ruiz G, & Wacher C. (2021). Microbial Interactions between Amylolytic and Non-Amylolytic Lactic Acid Bacteria Strains Isolated during the Fermentation of Pozol. Foods, 10(11), 2607.
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2

Real-Time PCR Analysis Protocol

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The methods for the real-time PCR analysis were described previously [61 (link),62 (link)]. A 7900 real-time PCR system (Applied Biosystems, Foster City, CA, USA) with HotStart-IT SYBR-Green qPCR Master Mix (Thermo Fisher Scientific, Waltham MA, USA) was used in this study. The primers used are shown in Table 1. The cycling profile for real-time PCR (50 cycles) was as follows: 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 60 s. The comparative threshold cycle (Ct) method (i.e., 2−ΔΔCt) was used to calculate fold amplification.
Park S.B., Yang Y., Bang S.I., Kim T.S, & Cho D. (2024). AESIS-1, a Rheumatoid Arthritis Therapeutic Peptide, Accelerates Wound Healing by Promoting Fibroblast Migration in a CXCR2-Dependent Manner. International Journal of Molecular Sciences, 25(7), 3937.
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3

Genome-wide DNA Methylation Analysis

Cited 1 time
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Genomic DNA was isolated using Purelink genomic DNA isolation kits (Invitrogen). For locus-wide bisulfite sequencing, CATCH-seq was performed as described12 (link), using BAC clone RP24-330J12 (BACPAC Resource Center, CHORI). For amplicon sequencing, bisulfite conversion was performed using the EpiTect Bisulfite Kit (Qiagen). Amplicons were prepared using Hot Start Ex-Taq Polymerase (TaKaRa) and the following primers: TSS-F: 5′-GGGGTATTTATTGTTTTGAGTAT-3′, TSS-R: 5′-TTTAATTTTTCAACTTCCCCAAC-3′, +1600-F: 5′-GGTTATTTGGAGTTTTTTTTTAG-3′, +1600-R: 5′-CTTCAATTCATAAACTTATTCCC-3′, and TA cloned using the Qiagen PCR cloning kit. Bisulfite analysis of Sanger sequenced clones was performed using QUMA40 (link). Oxidative bisulfite treatment was performed as previously described41 (link) and amplicons were analyzed as above using the following primers: +1400-F: 5′-AAGTGTTTAAAATGTGTTAATTATTG-3′, +1400-R: 5′-TTAAAAACAAAACTAAAAAAACCC-3′. T4-βGT-mediated 5mC- and 5hmC- sensitive restriction enzyme digest was performed using the EpiMARK 5-hmC and 5mC analysis kit according to the manufacturer’s instructions (New England Biolabs). Quantitative PCR was performed using HotStart-IT SYBR Green qPCR Master Mix (Affymetrix-USB) and a LightCycler 480 (Roche). Percent digestion was calculated using ΔΔCt.
Sellars M., Huh J.R., Day K., Issuree P.D., Galan C., Gobeil S., Absher D., Green M.R, & Littman D.R. (2015). Regulation of DNA methylation dictates Cd4 gene expression during development of helper and cytotoxic T cell lineages. Nature immunology, 16(7), 746-754.
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4

Temporal gene expression analysis of Nkx2.5-eGFP ESCs

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Nkx2.5-eGFP ESCs were seeded in 24-well plates (Corning Life Sciences) and treated with growth factors at day 1 or 2 of culture. At different time points after growth factor addition (i.e. day 0, 2, 4, 6 and 8), cells were lysed in TRIzol Reagent (Life Technologies) and total RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, the Netherlands). Reverse transcription was done with iScript cDNA Synthesis Kit (Bio-Rad, Hercules CA, USA). Gene expression levels were assayed using HotStart-IT SYBR Green qPCR Master Mix (Affymetrix, Santa Clara, CA, USA) and the primer pairs specified in Supplemental Table 2. PCR amplifications were performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Each condition was tested in three independent experiments using three samples per experiment.
Engels M.C., Rajarajan K., Feistritzer R., Sharma A., Nielsen U.B., Schalij M.J., de Vries A.A., Pijnappels D.A, & Wu S.M. (2014). IGF promotes Cardiac Lineage Induction In Vitro by Selective Expansion of Early Mesoderm. Stem cells (Dayton, Ohio), 32(6), 1493-1502.
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5

Quantitative RT-PCR for Muscle mRNA

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Total RNA was isolated from whole muscles using TRIzol Reagent (LifeTechnologies), following the manufacturer's protocol. cDNA was synthesized using SuperScript II Reverse Transcriptase, and qPCR performed using gene specific primers (see Supplementary Table for primers), the HotStart-IT SYBR green qPCR master mix (Affymetrix) and the QuantStudio 6 Flex Real Time PCR system (Applied Biosystems). Relative mRNA levels were determined after normalization to cyclophilin (Ppia, all ERR mKO comparisons) or 36B4 (Rpl0, used in data of Figure 1) mRNA as reference gene.
Wattez J.S., Eury E., Hazen B.C., Wade A., Chau S., Ou S.C., Russell A.P., Cho Y, & Kralli A. (2023). Loss of skeletal muscle estrogen-related receptors leads to severe exercise intolerance. Molecular Metabolism, 68, 101670.
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6

Genome-wide DNA Methylation Analysis

Cited 1 time
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Genomic DNA was isolated using Purelink genomic DNA isolation kits (Invitrogen). For locus-wide bisulfite sequencing, CATCH-seq was performed as described12 (link), using BAC clone RP24-330J12 (BACPAC Resource Center, CHORI). For amplicon sequencing, bisulfite conversion was performed using the EpiTect Bisulfite Kit (Qiagen). Amplicons were prepared using Hot Start Ex-Taq Polymerase (TaKaRa) and the following primers: TSS-F: 5′-GGGGTATTTATTGTTTTGAGTAT-3′, TSS-R: 5′-TTTAATTTTTCAACTTCCCCAAC-3′, +1600-F: 5′-GGTTATTTGGAGTTTTTTTTTAG-3′, +1600-R: 5′-CTTCAATTCATAAACTTATTCCC-3′, and TA cloned using the Qiagen PCR cloning kit. Bisulfite analysis of Sanger sequenced clones was performed using QUMA40 (link). Oxidative bisulfite treatment was performed as previously described41 (link) and amplicons were analyzed as above using the following primers: +1400-F: 5′-AAGTGTTTAAAATGTGTTAATTATTG-3′, +1400-R: 5′-TTAAAAACAAAACTAAAAAAACCC-3′. T4-βGT-mediated 5mC- and 5hmC- sensitive restriction enzyme digest was performed using the EpiMARK 5-hmC and 5mC analysis kit according to the manufacturer’s instructions (New England Biolabs). Quantitative PCR was performed using HotStart-IT SYBR Green qPCR Master Mix (Affymetrix-USB) and a LightCycler 480 (Roche). Percent digestion was calculated using ΔΔCt.
Sellars M., Huh J.R., Day K., Issuree P.D., Galan C., Gobeil S., Absher D., Green M.R, & Littman D.R. (2015). Regulation of DNA methylation dictates Cd4 gene expression during development of helper and cytotoxic T cell lineages. Nature immunology, 16(7), 746-754.
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7

Quantification of Fungal DNA Copy

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Total DNA was isolated from the selected A. oryzae transformants using E.Z.N.A. ® Fungal DNA Mini Kit (Omega Bio-tek, USA). DNA quantifications by realtime PCR were carried out with IQ5 realtime PCR system (Bio-Rad, USA). HotStart-IT SYBR Green qPCR Master Mix (Affymetrix, USA) and the primer pair pyrG-RT-F/pyrG-RT-R for the pyrG gene (Table 2) were included. The 2 -∆Ct formula was used for determining the phyA gene copy number, in which ∆Ct corresponds to Ct (in the strain of interest)-Ct (in the reference strain) (Schmittgen & Livak, 2008) .
Dung T.H., & Tran V. (2020). Heterologous phytase expression in the food filamentous fungus <i> Aspergillus oryzae </i>using the added rice husk cultivation model. ACADEMIA JOURNAL OF BIOLOGY, 42(2).
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8

Real-Time PCR Analysis Using Applied Biosystems

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Real-time PCR analysis was performed using the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The cDNA, appropriate primers, ROX dye and HotStart-IT SYBR Green qPCR Master Mix (USB Corporation, Cleveland, OH, USA) were incubated at 94 °C for 2 min, then for 45 cycles of 95 °C for 15 s and finally 60 °C for 1 min. Oligonucleotide primer sequences used are listed in Table 1.
Kim D.H, & Do M.S. (2015). BAFF knockout improves systemic inflammation via regulating adipose tissue distribution in high-fat diet-induced obesity. Experimental & Molecular Medicine, 47(1), e129-.
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9

Quantitative RT-PCR Analysis of Mouse Liver

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Total RNA from frozen liver samples of C57BL/6 and TLR2 KO mice was extracted using TRIzol reagent (Invitrogen, Burlington, Ont., Canada) and residual genomic DNA was removed with the Turbo DNA‐free kit (Ambion, Austin, TX). Cell culture RNA was extracted with the NucleoSpin® RNA II kit (Macherey‐Nagel GmbH & Co. KG, Düren, Germany). One microgram of RNA was retro‐transcribed into cDNA using the high‐capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Real‐time PCR amplification was carried out on 25 ng cDNA using the HotStart‐IT™ SYBR® Green qPCR Master Mix (USB Corporation, Cleveland, OH) on an ABI 7300 system (Applied Biosystems). Threshold cycle values (Ct) were collected and used for ‘ΔΔCt’ analysis. Specific primers for hypoxanthine phosphoribosyltransferase (HPRT), MHV‐nucleocapsid, TLR2, TLR3, TLR4, TLR7, MDA‐5, RIG‐1, IFN‐β, IL‐6, TNF‐α, IL‐33, Fgl‐2, CXCL1, CXCL10 and CCL2 were used (Table 1). The relative gene expression was normalized to HPRT as endogenous control and expressed as a ratio to gene expression in mock‐infected mice livers (arbitrarily taken as 1). The specificity of the PCR products was confirmed by melting curve analyses.
Bleau C., Burnette M., Filliol A., Piquet‐Pellorce C., Samson M, & Lamontagne L. (2016). Toll‐like receptor‐2 exacerbates murine acute viral hepatitis. Immunology, 149(2), 204-224.
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10

HUVEC miRNA and ASK1 Expression Analysis

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Total RNAs were extracted from HUVECs using a miRNeasy Mini Kit or RNeasy kit (QIAGEN). Quantitative real-time PCR (qRT-PCR) was performed with cDNA generated from 20 ng total RNA using a miRCURY LNATM Universal cDNA Synthesis kit and SYBR® Green Master Mix Kit (Exqion). MiR-19a primers were 5’-CCTCTG-TTAGTTTTGCATAGTTGC-3’ and 5’-CAGGCCACCATCAGTTTTG-3’; miR-20a primers were 5’-ACACTCCAGCTGGGTAAAGTGCTTATAGTGC-3’ and 5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTACCTGC-3’ (stem-loop reverse primer). qRT-PCR analysis of ASK1 expression was performed with cDNA generated from 250 ng total RNA using HotStart-IT® SYBR® Green qPCR Master Mix with a UDG (2×) Tested User FriendlyTM kit (USB Corporation). ASK1 primers were 5’-AGACATCTGGTCTCTGGGCTGTAC-3’ and 5’-AACATTCCCACCTTGAACATAGC-3’. The relative expression level was calculated by the 2-ΔΔCt method with the CT values normalized to 18S rRNA as the internal control for ASK1 and U6 snRNA as the internal control for miRNAs.
Jiang W.L., Zhang Y.F., Xia Q.Q., Zhu J., Yu X., Fan T, & Wang F. (2015). MicroRNA-19a regulates lipopolysaccharide-induced endothelial cell apoptosis through modulation of apoptosis signal-regulating kinase 1 expression. BMC Molecular Biology, 16, 11.
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