Quantitect sybr green master mix

Manufactured by Thermo Fisher Scientific

The QuantiTect SYBR Green Master Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) that contains SYBR Green I dye, HotStarTaq DNA Polymerase, dNTPs, and buffer components. It is designed for sensitive and reproducible quantification of DNA targets.

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Lab products found in correlation

M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Turbo dnase, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Dnase 1, by Qiagen (1 mentions) Superscript 3 first strand cdna synthesis, 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) QuantiTect SYBR Green mix (2 citations) QuantiTect SYBR green (2 citations)

4 protocols using quantitect sybr green master mix

RNA Isolation and qRT-PCR Analysis

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Total RNA was isolated from heads with TRIzol (Invitrogen) and treated with Turbo DNase (Invitrogen) to degrade contaminating DNA. For qRT-PCR analyses, cDNA was generated using MMLV Reverse transcriptase (Invitrogen) from 1 μg of total RNA, and then used as template for quantitative real-time PCR (qPCR) from duplicate samples of 10 ng cDNA with QuantiTect SYBR Green Master Mix using an Applied Biosystems StepOne Plus real time machine (ABI). Results were analyzed using the ΔΔCT method and normalized to Act5c as fold change relative to control. Oligonucleotide sequences are provided in (S1 Table).

Morton D.J., Jalloh B., Kim L., Kremsky I., Nair R.J., Nguyen K.B., Rounds J.C., Sterrett M.C., Brown B., Le T., Karkare M.C., McGaughey K.D., Sheng S., Leung S.W., Fasken M.B., Moberg K.H, & Corbett A.H. (2020). A Drosophila model of Pontocerebellar Hypoplasia reveals a critical role for the RNA exosome in neurons. PLoS Genetics, 16(7), e1008901.

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Quantitative Real-Time PCR Analysis of mRNA

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Total RNA was isolated from N2a cells using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Isolated RNA was treated with Turbo DNase (Invitrogen) to degrade contaminating DNA. For qRT-PCR analyses, cDNA was generated using the MMLV Reverse transcriptase (Invitrogen) from 1 μg of total RNA. Relative mRNA levels were measured by quantitative PCR analysis of duplicate samples of 15 ng cDNA with QuantiTect SYBR Green Master Mix using an Applied Biosystems StepOne Plus real time machine (ABI). Results were analyzed using the ΔΔCT method and normalized to 18S rRNA. Oligonucleotide sequences are provided in Supplementary Table S2.

Morris K.J, & Corbett A.H. (2018). The polyadenosine RNA-binding protein ZC3H14 interacts with the THO complex and coordinately regulates the processing of neuronal transcripts. Nucleic Acids Research, 46(13), 6561-6575.

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Quantitative RT-PCR Methodology

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For qRT-PCR analyses, 1 μg of total RNA was transcribed to cDNA as described above. Relative mRNA levels were measured by quantitative PCR analysis of triplicate samples of 10 ng of cDNA with QuantiTect SYBR Green Master Mix using an Applied Biosystems real time machine (ABI). Fold change was calculated using the ΔΔCt method, by normalizing to the HPRT transcript for the first ΔCt and to either control IgG IP samples (siRNA mediated knockdown followed by endogenous protein RNA-immunoprecipitation) or primers sets for the coding sequence (siRNA mediated knockdown affect on polyadenylation site usage and retained intron) for the second ΔCt. Statistical significance was determined using Student's t-test. A list of primers used for these analyses is shown in Supplemental Table S1.

Banerjee A., Vest K.E., Pavlath G.K, & Corbett A.H. (2017). Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing. Nucleic Acids Research, 45(18), 10706-10725.

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RNA Extraction and Quantitative PCR Analysis

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Total RNA was isolated from adult tissues with TRIzol (Invitrogen) and treated with DNase I (QIAGEN). For RT-PCR, cDNA was generated using SuperScript III First Strand cDNA Synthesis (Invitrogen) from 2 μg of total RNA, and PCR products were resolved and imaged on 2% agarose gels (Bio-Rad image). Quantitative real-time PCR (qPCR) reactions were carried out in biological triplicate with QuantiTect SYBR Green Master Mix using an Applied Biosystems StepOne Plus real-time machine (ABI). Results were analyzed using the ΔΔCT method, normalized as indicated (e.g., to Act5C), and plotted as fold-change relative to control.

Jalloh B., Lancaster C.L., Rounds J.C., Brown B.E., Leung S.W., Banerjee A., Morton D.J., Bienkowski R.S., Fasken M.B., Kremsky I.J., Tegowski M., Meyer K., Corbett A, & Moberg K. (2023). The Drosophila Nab2 RNA binding protein inhibits m6A methylation and male-specific splicing of Sex lethal transcript in female neuronal tissue. eLife, 12, e64904.

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