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Fast sybr green pcr master mix

Manufactured by Thermo Fisher Scientific
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Fast SYBR Green PCR Master Mix is a ready-to-use solution for real-time PCR amplification and detection using SYBR Green I dye. It contains all the necessary components, including a hot-start DNA polymerase, dNTPs, and SYBR Green I, to perform real-time PCR reactions.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (41 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (38 mentions) Rneasy mini kit, by Qiagen (37 mentions) 7500 fast real time pcr system, by Thermo Fisher Scientific (28 mentions) Trizol, by Thermo Fisher Scientific (20 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (15 mentions) Dnase 1, by Thermo Fisher Scientific (11 mentions) Reverse transcription system, by Promega (10 mentions) Rneasy kit, by Qiagen (9 mentions) Random primer, by Thermo Fisher Scientific (9 mentions) Nanodrop system, by Thermo Fisher Scientific (9 mentions) Total rna purification kit, by Merck Group (7 mentions) Rneasy micro kit, by Qiagen (7 mentions) Abi 7900 fast real time pcr system, by Thermo Fisher Scientific (7 mentions) Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (7 mentions) Stepone software v2, by Thermo Fisher Scientific (7 mentions) M mlv reverse transcriptase, by Thermo Fisher Scientific (6 mentions) Steponeplus, by Thermo Fisher Scientific (6 mentions) Abi prism 7000, by Thermo Fisher Scientific (6 mentions) Iscript cdna synthesis kit, by Bio-Rad (6 mentions)

231 protocols using fast sybr green pcr master mix

1

Quantifying Bt mRNA Levels by qPCR

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mRNA was prepared from 1.0 mL of Bt cell culture pre-treated with RNA protect (Qiagen) using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. Contaminating DNA was removed using on-column DNase treatment (Qiagen) during purification according to the manufacturer’s instructions. cDNA was synthesized from 1.0 μg of RNA using Superscript VILO IV master mix (ThermoFisher) according to the manufacturer’s instructions. mRNA levels were measured by quantification of cDNA using Fast SYBR Green PCR Master Mix (Applied Biosystems) and primers listed in Table S2 using a QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems, Waltham, Massachusetts, USA). Data were normalized to 16s ribosomal RNA from 1,000-fold diluted cDNA as previously described26 (link). qPCR primers are listed in Table S2.
Pearce V.H., Groisman E.A, & Townsend GE I.I. (2023). Dietary sugars silence the master regulator of carbohydrate utilization in human gut Bacteroides species. Gut Microbes, 15(1), 2221484.
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2

RNA Isolation and RT-PCR Analysis of AMs

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mRNA from cultured AMs, in vivo sorted AMs or homogenates from individual lungs as indicated in the text was isolated with Total RNA purification kit (Sigma) and treated with DNase I (Invitrogen). Random primers (Invitrogen) and Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen) were used to synthesize first-strand cDNAs from equivalent amounts of RNA from each sample. RT-PCR was performed with Fast SYBR Green PCR Master Mix (Applied Biosystems). RT-PCR was conducted in duplicates in QuantStudio3 (Applied Bioscience). Data were generated with the comparative threshold cycle (Delta CT) method by normalizing to hypoxanthine phosphoribosyltransferase (HPRT). Sequences of primers used in the studies are provided in the Table S1.
Zhu B., Wu Y., Huang S., Zhang R., Son Y.M., Li C., Cheon I.S., Gao X., Wang M., Chen Y., Zhou X., Nguyen Q., Phan A.T., Behl S., Taketo M.M., Mack M., Shapiro V.S., Zeng H., Ebihara H., Mullon J.J., Edell E.S., Reisenauer J.S., Demirel N., Kern R.M., Chakraborty R., Cui W., Kaplan M.H., Zhou X., Goldrath A.W, & Sun J. (2021). Uncoupling of macrophage inflammation from self-renewal modulates host recovery from respiratory viral infection. Immunity, 54(6), 1200-1218.e9.
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3

Quantitative PCR Analysis of Klf4 Knockout

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cDNA from Klf4-null and wild-type littermates were produced with random hexamers using the High Capacity cDNA Archive kit (Applied Biosystems). cDNA products were diluted to 100 ng total RNA input. Sequences of the transcript of interest were loaded into Primer Express software (Applied Biosystems). Amplicon lengths were between 75 and 125 bp. The qPCR was performed with the FAST SYBR Green PCR Master Mix (Applied Biosystems) on an ABI StepOne Plus Sequence Detection System (Applied Biosystems). All runs were normalised to 18s RNA. Three biological replicates were prepared for each gene target and three technical replicates were performed for each biological replicate. Gene expression was represented as relative quantity against the negative control which used water as the template (noted as “Relative Quantity vs. H2O” in Figs).
The results of Real-Time PCR were analyzed and graphed by ABI StepOne Plus Sequence Detection System (Applied Biosystems). The expression data was statistically analyzed using a one-tailed Students’ T-test with p<0.05 as significant.
Zhang P., Ha T., Larouche M., Swanson D, & Goldowitz D. (2015). Kruppel-Like Factor 4 Regulates Granule Cell Pax6 Expression and Cell Proliferation in Early Cerebellar Development. PLoS ONE, 10(7), e0134390.
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4

Quantifying Gene Expression in Drosophila

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Total RNA was extracted from 20 flies using Trizol (Invitrogen Life Technology) according to the manufacturer's instructions. The RNA was used directly for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) followed by DNase I treatment (Invitrogen Life Technology). Real-time PCR using Fast SYBR Green PCR Master Mix (Applied Biosystems) was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems). Primer used for RT-PCR: SMC6 (5′-gatgaagtgaaccggaagtttattac-3′ and 5′-tagcctcgaccttcgtatcc-3′), CG6204 (5′-ctttgaacgactcattgtggc-3′ and 5′-gatgtcctcgtattccttcactg-3′), and RP49 (5′-gtcgccgcttcaagggacagta-3′ and 5′-gcgatctcgccgcagtaaac-3′).
Tran M., Tsarouhas V, & Kegel A. (2016). Early development of Drosophila embryos requires Smc5/6 function during oogenesis. Biology Open, 5(7), 928-941.
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5

Adiponectin Regulation in Murine Adipocytes

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Pioglitazone was obtained from the NCI Chemical Repository. Mouse recombinant TNF-α, and ELISA kits for MCP-1 and adiponectin were obtained from R&D Systems. RNeasy mini kits, real-time PCR mouse primers for CD68 (Cat # QT00254051), MCP-1 (Cat # QT00167832), TGF-β (Cat # QT00145250), GAPDH (Cat # QT01658692), adiponectin (Cat # QT01048047), AdipoR1 (Cat # QT00154217), AdipoR2 (Cat # QT00165326) and siRNA for adiponectin were purchased from Qiagen. TNF-α primers were obtained from Sigma (17 (link)). Non-targeting siRNA, AdipoR2 siRNA and DharmaFECT4 were obtained from Dharmacon. Murine leukemia virus reverse transcriptase, RNase inhibitor, oligo (dT)16, and Fast SYBR green PCR master mix were obtained from Applied Biosystems. Insulin, IBMX and dexamethasone were obtained from Sigma.
Miyazawa M., Subbaramaiah K., Bhardwaj P., Zhou X.K., Wang H., Falcone D.J., Giri D.D, & Dannenberg A.J. (2017). Pioglitazone Inhibits Periprostatic White Adipose Tissue Inflammation in Obese Mice. Cancer prevention research (Philadelphia, Pa.), 11(4), 215-226.
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6

Quantitative Gene Expression Analysis

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Total RNA was isolated using the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. The purified RNA was quantified using a Nanodrop machine (NanoDrop Technologies). Complementary DNA (cDNA) was synthesized using High Capacity RNA-to-cDNA Master Mix (Applied Biosystems). The mRNA amount of the STM14_1977, ycjE, csgD, proV, pdeG, rclC, and yjhB genes was determined by quantification of cDNA using Fast SYBR Green PCR Master Mix (Applied Biosystems) and appropriate primers (ycjE: 16675/16676; STM14_1977: 15973/15974; csgD: 17620/17621; proV: 17618/17619; pdeG: 17622/17623; rclC: 17624/17625; yjhB: 17626/17627; ompA: W3961/W3962; rrs: 3203/3204) and monitored using a QuantStudio 6 machine (Applied Biosystems). Data were normalized to the levels of 16S ribosomal RNA (rrs) or ompA.
Choi J., Schmukler M, & Groisman E.A. (2022). Degradation of gene silencer is essential for expression of foreign genes and bacterial colonization of the mammalian gut. Proceedings of the National Academy of Sciences of the United States of America, 119(40), e2210239119.
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7

Quantifying mRNA Levels via RT-qPCR

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RNA was isolated using RNA-STAT60 (Tel-Test) according to the manufacturer’s instructions and subjected to DNAse I (Ambion) digestion to remove residual DNA. Purified RNA was then reverse transcribed with random hexamer and oligo-dT (Yuan et al., 2015 (link)) (Applied Biosystems) and amplified on a 7500 Fast Real-Time PCR system using Fast SYBR Green PCR Master Mix (Applied Biosystems). The sequences for primers are included in Appendix 1—table 1. mRNA levels were calculated based on the difference of threshold Ct values in the target gene and average Ct values of 18s, Actb, B2m, and Hprt in the same sample.
Yang X., Chang H.C., Tatekoshi Y., Mahmoodzadeh A., Balibegloo M., Najafi Z., Wu R., Chen C., Sato T., Shapiro J, & Ardehali H. (2023). SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury. eLife, 12, e85571.
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8

Quantitative Gene Expression Analysis

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Following treatments, total RNA was extracted from NP cells (5 × 105 cells/plate) using RNAeasy micro columns (Qiagen). Before elution from the column, RNA was treated with RNase free DNAse I. 2 μg of total DNA free-RNA was used to synthesize cDNA using SuperScipt III cDNA synthesis kit (Invitrogen). Reactions were set up in triplicate in 96-well plates using 1 μl cDNA with Fast SYBR Green PCR Master Mix (Applied Biosystems) to which gene-specific forward and reverse PCR primers were added. Each set of samples included a template-free control. PCR reactions were performed in a StepOnePlus real-time PCR system (Applied Biosystems) according to the manufacturer’s instructions. Expression of the gene of interest was first normalized by housekeeping gene Hprt1 and then data presented as relative to the corresponding control group. All primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).
Tian Y., Yuan W., Li J., Wang H., Hunt M.G., Liu C., Shapiro I.M, & Risbud M.V. (2015). TGFβ Regulates Galectin-3 Expression through Canonical Smad3 Signaling Pathway in Nucleus Pulposus Cells: Implications in Intervertebral Disc Degeneration. Matrix biology : journal of the International Society for Matrix Biology, 50, 39-52.
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9

Quantifying Fmo3 mRNA in Mouse Liver

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Total RNA was extracted from mouse liver samples using TRIzol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Total RNAwas then reverse-transcribed into cDNA using an M-MLV RT kit (Invitrogen, Carlsbad, CA). Fmo3 mRNA expression was quantified by the ΔΔCT method and normalized to two housekeeping genes, β-actin and ribosomal protein S18. Data presented were normalized to β-actin. Primer pairs were synthesized by Integrated DNA Technologies (Coralville, IA) and are as follow: Fmo3 forward: 5′-GGA AGA GTT GGT GAA GAC CG-3′, reverse: 5′-CCC ACA TGC TTT GAG AGG AG-3′. Amplification was performed using an Applied Biosystems 7500 Fast Real-Time PCR System. Amplification was carried out in a 20μL reaction volume containing 8 μL diluted cDNA, Fast SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 1μM of each primer.
Rudraiah S., Moscovitz J.E., Donepudi A.C., Campion S.N., Slitt A.L., Aleksunes L.M, & Manautou J.E. (2014). Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice. Toxicology, 325, 85-95.
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10

RNA Extraction and qPCR Protocol for Ccr9 Expression

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Tissue RNA was extracted using the RNeasy Mini Kit (QIAGEN, Germantown, MD) per manufacturer’s instructions. Reverse transcription was carried out with a high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA) per manual. Real-time PCR was performed using the Fast SYBR Green PCR Master Mix (Applied Biosystems) in Applied Biosystems 7500 Real-Time PCR System. Relative fold change was calculated by the ΔΔCT method with 18s ribosomal RNA as an internal control. Primers were synthesized by Integrated DNA Technologies (Coralville, Iowa) as sequence follows: 18s (5′-GTA ACC CGT TGA ACC CCA TT-3’and 5′-CCA TCC AAT CGG TAG TAG CG-3′); Ccr9 [46 (link)] (5′-CAA TCT GGG ATG AGC CTA AAC AAC 3′ and 5′-ACC AAA AAC CAA CTG CTG CG-3′).
Li M., Bou-Dargham M.J., Yu J., Etwebi Z., Sun H, & Chen Y.H. (2021). TIPE polarity proteins are required for mucosal deployment of T lymphocytes and mucosal defense against bacterial infection. Molecular Biomedicine, 2, 41.
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