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Quantifast sybr green reagent

Manufactured by Qiagen
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The Quantifast SYBR Green reagent is a real-time PCR assay designed for the quantitative detection of DNA sequences. It utilizes SYBR Green dye technology to facilitate the monitoring of DNA amplification during the PCR process.

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Lab products found in correlation

Cfx96 real time analyzer, by Bio-Rad (3 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions) Ab1791, by Abcam (1 mentions) Protein g dynabead, by Thermo Fisher Scientific (1 mentions) Steponeplus, by Thermo Fisher Scientific (1 mentions) Anti p53 do 1, by Santa Cruz Biotechnology (1 mentions) Protein a g dynabeads, by Thermo Fisher Scientific (1 mentions) Anti histone h3 acetyl k9, by Abcam (1 mentions) Anti acetyl histone h4, by Merck Group (1 mentions)

3 protocols using quantifast sybr green reagent

1

Quantifying TRIM37 Expression by qPCR

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Total RNA was extracted using the RNeasy Plus Mini kit (Qiagen) and reverse transcription was performed using the iScript™ cDNA Synthesis Kit (Bio-Rad) following manufacturer’s protocol. TRIM37 transcripts were measured by qPCR in triplicate on a CFX96 Real-Time Analyzer (Bio-Rad) using Quantifast SYBR Green reagent (QIAGEN), normalised to reference gene HBMS and quantified using the ΔΔCt method to obtain relative expression. Thermocycling conditions were set as follows: 1 cycle (95 °C for 5mins), 40 cycles (95 °C for 15s, 58 °C for 60s). Primer sequences: TRIM37 forward (5’-TCAGCTGTATTAGGCGCTGG-3’), TRIM37_reverse (5’-ACTTCTTCTGCCCAACGACA-3’), HBMS_forward (5’- GCCCTGGAGAAGAATGAA-3’), HBMS_reverse (5’-GGTGAAAGACAACAGCATC-3’).
Yeow Z.Y., Lambrus B.G., Marlow R., Zhan K.H., Durin M.A., Evans L.T., Scott P.M., Phan T., Park E., Ruiz L.A., Moralli D., Knight E.G., Badder L.M., Novo D., Haider S., Green C.M., Tutt A.N., Lord C.J., Chapman J.R, & Holland A.J. (2020). Exploiting TRIM37-driven centrosome dysfunction to kill breast cancer cells. Nature, 585(7825), 447-452.
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2

ChIP-qPCR analysis of CH12-F3 cells

Cited 1 time
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Each ChIP was performed from chromatin prepared from ~107 CH12-F3 cells stimulated for 30 h with agonist CD40 antibody, IL-4, and TGF-β as previously described13 (link). Each ChIP was performed using 30–50 μg CH12-F3 chromatin using RPA34-20 (3 μg; Ab-3, Calbiochem) or Anti-Histone H3 (2 μg; ab1791, Abcam) coupled to 25 μl Protein-G Dynabeads® (Life Technologies, 10003D). Relative quantities of ChIP-enriched DNA were calculated relative to total input chromatin by qPCR in triplicate on CFX96 Real-Time Analyzer (Bio-Rad) or StepOnePlus (Applied Biosystems) instruments using Quantifast SYBR Green reagent (QIAGEN) and locus-specific primer pairs (Extended Data Table 4b).
Ghezraoui H., Oliveira C., Becker J.R., Bilham K., Moralli D., Anzilotti C., Fischer R., Deobagkar-Lele M., Sanchiz-Calvo M., Fueyo-Marcos E., Bonham S., Kessler B.M., Rottenberg S., Cornall R.J., Green C.M, & Chapman J.R. (2018). 53BP1 cooperation with the REV7-Shieldin complex underpins DNA structure-specific NHEJ. Nature, 560(7716), 122-127.
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3

ChIP-qPCR Protocol for Chromatin Immunoprecipitation

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ChIP experiments were performed from 30–50 μg MCF-7 chromatin essentially as previously described (Chapman et al., 2013 (link)). Briefly, chromatin was immunoprecipitated using 3 μg anti-p53 (DO-1, Santa Cruz), 1.5 μg anti-RNAP2 CTD (phospho-S5; Clone 4H8), 2.5μg Anti-Histone H3 (acetyl K9) (ab4441, Abcam), or 2.5 μg anti-acetyl-histone H4 (06-598, Millipore) antibody coupled to 25 μl Protein-A/G Dynabeads (Life Technologies). DNA quantities recovered in control IgG ChIP experiments were consistently below the detectable range. Relative quantities of ChIP-enriched DNA were calculated relative to total input chromatin by qPCR in triplicate on a CFX96 Real-Time Analyzer (Bio-Rad) using Quantifast SYBR Green reagent (QIAGEN) and locus-specific primer pairs (sequences in Supplemental Experimental Procedures).
Cuella-Martin R., Oliveira C., Lockstone H.E., Snellenberg S., Grolmusova N, & Chapman J.R. (2016). 53BP1 Integrates DNA Repair and p53-Dependent Cell Fate Decisions via Distinct Mechanisms. Molecular Cell, 64(1), 51-64.
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