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Fast start sybr green 1

Manufactured by Roche
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Fast Start SYBR Green I is a real-time PCR reagent designed for quantitative gene expression analysis. It contains SYBR Green I, a DNA-binding fluorescent dye, and a thermostable DNA polymerase that enables fast and efficient amplification of target sequences.

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Lab products found in correlation

High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions) Lightcycler 2, by Roche (2 mentions) 480 sybr green 1, by Roche (2 mentions) Turbo dna free, by Thermo Fisher Scientific (2 mentions) Lightcycler 480 2, by Roche (2 mentions) Rneasy mini kit, by Qiagen (1 mentions) Iscript, by Bio-Rad (1 mentions) Dnase 1 digestion, by Qiagen (1 mentions) Lightcycler 96 system, by Roche (1 mentions) Lightcycler 480, by Roche (1 mentions)

Close spelling variants of this product

Fast-Start™ DNA Master SYBR Green I real-time PCR kit Fast Start SYBR Green I kit Fast Start SYBR Green I PCR mix LC Fast start DNA Master SYBR Green I Mix LightCycler-Fast Start DNA Master SYBR Green I Mix Fast Start DNA MasterPLUS SYBR Green I reaction mix Light Cycler Fast Start SYBR Green I Master Mix Fast SYBR Green SYBR Green I SYBR Green

4 protocols using fast start sybr green 1

1

Achilles Tendon RNA Extraction and qRT-PCR

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Achilles tendons were excised from the calcaneus, immediately distal to the tendon-muscular-junction. RNA was extracted with Trizol/choroform and purified with the RNeasy mini kit with on-column DNase I digestion (Qiagen). qRT-PCR was performed on the LightCycler® 96 System (Roche) using gene-specific primers and FastStart SYBR Green I (Roche) following cDNA synthesis with iScript (Bio-Rad). The sequences of primers are listed in Supplementary Table 4.
Lim J., Munivez E., Jiang M.M., Song I.W., Gannon F., Keene D.R., Schweitzer R., Lee B.H, & Joeng K.S. (2017). mTORC1 Signaling is a Critical Regulator of Postnatal Tendon Development. Scientific Reports, 7, 17175.
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2

RNA Extraction, cDNA Synthesis, and qPCR

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Total RNA was isolated using a standard TriReagent protocol and treated with DNAse (TURBO DNA-free, AM1907M, Ambion). An aliquot was used for cDNA synthesis using a High Capacity RNA-to-cDNA kit (Applied Biosystems, UK) containing Oligo-dT and random primers. Real-time PCR was performed using Roche Fast Start SYBR Green I on a Lightcycler2 with Lightcycler 3.5 software or using Roche 480 SYBR Green I on a Lightcycler480II with Lightcycler 1.5.62 software. DNA amplification was for 35 cycles with an initial 10 min at 95 °C followed by 10 s at 95 °C, 6 s at 55 °C, and 14 s at 72 °C. Primers were used at 0.5 μmole/L. Sequences of PCR primer are specified in Table 1. The specificity of PCR was verified by reactions without RT (-RT) and by melt-curve analysis. PCR cycle crossing-points (CP) were determined by fit-points methodology. Relative abundance of target RNA was calculated from (E18SCp)/(EtargetCp). PCR products were electrophoresed on 2% agarose gels containing ethidium bromide. All quantitative PCR reactions were performed in duplicate and the data averaged to generate one value per experiment. On Fig. 1D, data are presented as Relative abundance. On Figs 2–6, data are presented after normalization to the Control group mean value.
Rode B., Yuldasheva N.Y., Baxter P.D., Sedo A., Ainscough J.F., Shires M., Kearney M.T., Bailey M.A., Wheatcroft S.B, & Beech D.J. (2019). TRPC5 ion channel permeation promotes weight gain in hypercholesterolaemic mice. Scientific Reports, 9, 773.
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3

Quantitative Real-Time PCR Protocol

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Total RNA was isolated using a standard TriReagent protocol and treated with DNAse (TURBO DNA-free, AM1907M, Ambion). An aliquot was used for cDNA synthesis using a High Capacity RNA-to-cDNA kit (Applied Biosystems, UK) containing Oligo-dT and random primers. Real-time PCR was performed using Roche Fast Start SYBR Green I on a Lightcycler2 with Lightcycler 3.5 software or using Roche 480 SYBR Green I on a Lightcycler480II with Lightcycler 1.5.62 software. DNA amplification was for 35 cycles with an initial 10 min at 95 °C followed by 10 s at 95 °C, 6 s at 55 °C and 14 s at 72 °C. Primers were used at 0.5 μM. Sequences of PCR primers are specified in Supplementary Table 1. The specificity of PCR was verified by reactions without RT (-RT) and by melt-curve analysis. PCR cycle crossing-points (CP) were determined by fit-points methodology. Relative abundance of target RNA was calculated from (E18sCp)/(EtargetCp). All quantitative PCR reactions were performed in duplicate and the data averaged to generate one value per experiment.
Rode B., Shi J., Endesh N., Drinkhill M.J., Webster P.J., Lotteau S.J., Bailey M.A., Yuldasheva N.Y., Ludlow M.J., Cubbon R.M., Li J., Futers T.S., Morley L., Gaunt H.J., Marszalek K., Viswambharan H., Cuthbertson K., Baxter P.D., Foster R., Sukumar P., Weightman A., Calaghan S.C., Wheatcroft S.B., Kearney M.T, & Beech D.J. (2017). Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance. Nature Communications, 8, 350.
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4

Quantitative Analysis of CypA mRNA

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CypA mRNA levels were analysed by SYBR green qPCR using the LightCycler 480 (Roche). Each PCR reaction consisted of 3 mmol/µl MgCl2, the respective primers at 0.5 pmol/µl, 1 µl Fast Start SYBR Green I (Roche), 1 µg cDNA and water to make the total reaction volume to 10µl. CypA cDNA was detected using the following primer set, CypA-F: 5’-GTCAACCCCACCGTGTTCTTC-3’ and CypA-R: 5’-TTTCTGCTGTCTTTGGGACCTTG-3’. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) determined to be the most suitable reference gene based on PCR efficiency was used to correct for differences in the cDNA input, GAPDH cDNA was detected using the following primer ser: GAPDH-F: 5’-GTCAACCCCACCGTGTTCTTC-3’ and GAPDH-R: 5’-TTTCTGCTGTCTTTGGGACCTTG-3’. SYBR green qPCR was performed using the following program on the LightCycler 480: (1) preincubation: 95°C for 5 min; (2) amplification: 45 cycles of 95°C for 15 sec, 60°C for 15 sec, 72°C for 15 sec; (3) melting curve: 95°C for 5 sec, 65°C for 1 min, 97°C for 0 sec with a temperature transition rate of 0.11°C/sec. To confirm amplification specificity, the PCR products were subjected to a melting curve analysis. Serial dilutions of cDNA from total RNA were performed for each target gene to create standard curves for quantitative analysis.
Madlala P., Singh R., An P., Werner L., Mlisana K., Abdool Karim S.S., Winkler C.A, & Ndung’u T. (2016). Association of polymorphisms in the regulatory region of the cyclophilin A gene (PPIA) with gene expression and HIV/AIDS disease progression. Journal of acquired immune deficiency syndromes (1999), 72(5), 465-473.
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