Fluorescein 12 utp

Manufactured by Merck Group

Sourced in Canada, United States

Fluorescein-12-UTP is a nucleotide analog used in various molecular biology and biochemistry applications. It contains a fluorescein dye molecule covalently attached to the uridine triphosphate (UTP) nucleotide. This modification allows the labeled UTP to be incorporated into RNA during in vitro transcription or other nucleic acid synthesis reactions, enabling the detection and visualization of the resulting RNA products.

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Lab products found in correlation

Nanodrop 2000, by Thermo Fisher Scientific (4 mentions) 5 end labeling kit, by Vector Laboratories (2 mentions) Ir800 dye maleimide probe, by LI COR (2 mentions) Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (2 mentions) Rna clean concentrator kit, by Zymo Research (2 mentions) Dnase 1, by Zymo Research (2 mentions) Dig 11 utp, by Merck Group (2 mentions) Pcrii vector, by Thermo Fisher Scientific (1 mentions) Strataclone, by Agilent Technologies (1 mentions) Digoxigenin 11 utp, by Merck Group (1 mentions) Nucleospin rna clean up kit, by Macherey-Nagel (1 mentions) Dnp 11 utp, by PerkinElmer (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Yeast trna, by Merck Group (1 mentions) Azure c600 imaging system, by Azure Biosystems (1 mentions) Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (1 mentions) 2 4 6 tri 2 pyridyl s triazine, by Merck Group (1 mentions) 2 2 azino bis, by Merck Group (1 mentions) Alcalase 2.4 l, by Novozymes (1 mentions) Thermostable inorganic pyrophosphatase, by New England Biolabs (1 mentions)

7 protocols using fluorescein 12 utp

CRISPR crRNA and Substrate RNA Synthesis

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DNA oligo templates for crRNA and substrate RNA in vitro transcription were ordered from IDT (Tables S1b). Templates for crRNAs were annealed to a short T7 primer (final concentrations 4µM) and incubated with T7 RNA polymerase overnight at 37°C using the HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs). Annealing was performed by incubating T7 primer with templates for 2 minutes at 95°C foll owed by a −5°C/s ramp down to 23°C. Templates for substrate RNA were PCR amplified to yield dsDNA and then incubated with T7 RNA polymerase at 37°C overnight using the same T7 Quick High Yield RNA Synthesis kit. After in vitro transcription, samples were treated with DNase I (Zymo Research) and then purified using RNA Clean & Concentrator kit (Zymo Research).
5’ end labeling was accomplished using the 5’ end labeling kit (VectorLabs) and with a IR800 dye-maleimide probe (LI-COR Biosciences). Body labeling of RNA was performed during in vitro transcription using the HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs). The in vitro transcription reactions contained 2.5 mM Fluorescein-12-UTP (Sigma Aldrich). Labeled RNA was purified to remove excess dyes using RNA Clean & Concentrator kit (Zymo Research). The RNA concentration was measured on Nanodrop 2000 (Thermo Fisher).

Yan W.X., Chong S., Zhang H., Makarova K.S., Koonin E.V., Cheng D.R, & Scott D.A. (2018). Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL domain-containing accessory protein. Molecular cell, 70(2), 327-339.e5.

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Cloning and Probing of Vertebrate TH and TPH Genes

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Following the general formatting of gene symbols, chicken genes will be abbreviated with upper‐case letters (e.g., TH1), while those in Xenopus and zebrafish will be abbreviated with lower‐case letters (e.g., th1). In order to refer to the homologous genes throughout the three species or throughout vertebrates, we will use upper‐case (e.g., TH1) in this article.
Chicken and Xenopus TH and TPH genes were cloned into pCRII Vector (Invitrogen/Thermo Fisher Scientific Inc.) or StrataClone (Agilent Technologies, Santa Clara, CA), after PCR amplification of the transcripts using specific primers (Table 2). Zebrafish th2 and tph1a had already been used in previous publications (Bellipanni, Rink, & Bally‐Cuif, 2002; Yamamoto et al., 2010, 2011). Antisense and sense RNA probes were synthesized by in vitro transcription using T3, T7, or Sp6 RNA polymerase (Promega, Madison, WI) and labeled with fluorescein‐12‐UTP or digoxigenin‐11‐UTP (Sigma‐Aldrich Co. LLC./Roche). Probes were purified using Nucleospin RNA clean‐UP kit (Macherey‐Nagel, Hoerdt, France) and analyzed by gel electrophoresis to confirm the size.

Xavier A.L., Fontaine R., Bloch S., Affaticati P., Jenett A., Demarque M., Vernier P, & Yamamoto K. (2017). Comparative analysis of monoaminergic cerebrospinal fluid‐contacting cells in Osteichthyes (bony vertebrates). The Journal of Comparative Neurology, 525(9), 2265-2283.

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Whole-mount in situ Hybridization Protocol

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Whole-mount in situ hybridizations were performed as described elsewhere (Pearson et al., 2009 (link)). Hybridized RNA probes were labeled with DIG-11-UTP (Sigma), Fluorescein-12-UTP (Sigma) or DNP-11-UTP (Perkin Elmer) and purified as described (Lapan and Reddien, 2011 (link)). Tyramide was generated by conjugation of succinimidyl esters of rhodamine, FITC, and AMCA with tyramide-HCL (Sigma) (Hopman et al., 1998 (link)). For horseradish peroxidase enzyme inactivation, animals were incubated in 154 mmol/L sodium azide for 2 h (King and Newmark, 2013 (link); van Wolfswinkel et al., 2014 (link)). Animals were counterstained with DAPI (Sigma, 3 μg/mL in PBSTx) for 1 h and mounted for imaging.

Han X.S., Wang C., Guo F.H., Huang S., Qin Y.W., Zhao X.X, & Jing Q. (2018). Neoblast-enriched zinc finger protein FIR1 triggers local proliferation during planarian regeneration. Protein & Cell, 10(1), 43-59.

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CRISPR crRNA and Substrate RNA Synthesis

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CBP Protein-RNA Interaction Assay

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Mobility shift assays were conducted with synthetic Fluorescein-labeled ∼150-base RNA oligonucleotides and specified concentrations of recombinant CBP HAT domain (Sigma, 1319–1710) and CBP bromodomain (Abcam, ab198130). Oligonucleotides were synthesized using acRNA-containing Display plasmids as template DNA and the HiScribe T7 High Yield RNA synthesis kit (NEB) with Fluorescein-12-UTP (Sigma). RNA oligonucleotides (eRNA1 46.11 ng, eRNA3 47.19 ng, ctrl RNA 46.51 ng for 20 nM final concentrations; up to 5 μM for competition assay) were heated to 95°C for 5min and refolded at room temperature prior to incubation with CBP protein (0–0.6 M) in REMSA buffer (20 mM HEPES, 40 mM KCl, 1 mM EDTA, 0.2 mM DTT, 0.1 mg ml^–1 BSA, 0.1% Tween-20 and 20% Glycerol, and 0.1 mg/ml yeast tRNA (Sigma)) for 1 h at 37°C. REMSA was performed using 1% agarose gel electrophoresis in 0.5× TBE. Electrophoretic mobility of fluorescein-labeled RNA was assayed using fluorescence imaging on the Azure c600 Imaging System (Azure biosystems). RNA–CBP complex formation was quantified as the Fluorescein signal intensity appearing at the higher band (corresponding to CBP-bound RNA with lower electrophoretic mobility) divided by the total signal intensity (bound RNA plus free probe).

Carullo N.V., Phillips III R.A., Simon R.C., Soto S.A., Hinds J.E., Salisbury A.J., Revanna J.S., Bunner K.D., Ianov L., Sultan F.A., Savell K.E., Gersbach C.A, & Day J.J. (2020). Enhancer RNAs predict enhancer–gene regulatory links and are critical for enhancer function in neuronal systems. Nucleic Acids Research, 48(17), 9550-9570.

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Antioxidant Capacity Measurement Protocols

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The reagents 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), 6-hydroxy-2,5,7,8-tetramethylchromo-2-carboxylic acid (Trolox), 3-(2-pyridyl), 2, 2′-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) and Fluorescein-12-UTP were obtained from Sigma-Aldrich (Oakville, Ontario, Canada) and 2,4,6-Tri-2-pyridyl-s-triazine (TPTZ) was supplied by Merck (Darmstadt, Germany). Alcalase® 2.4 L (commercial protease obtained from fermentation of Bacillus licheniformis, non-specific serine endopeptidase) was supplied by Novozymes (Bagsværd, Denmark). All reagents implemented in this study were analytical grade.

Gaviria G Y.S, & Zapata M J.E. (2023). Optimization and scale up of the enzymatic hydrolysis of Californian red worm protein (Eiseniafoetida). Heliyon, 9(5), e16165.

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Probe Synthesis for In Situ Hybridization

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Genes of interest were PCR-amplified from cDNA and cloned into pJC53.2 as previously described [Citation9] . The DNA template for probe synthesis was generated by PCR amplification using T7 primers and purified using a DNA clean and concentrator kit (Zymo Research, CA, USA). Antisense RNA probes were synthesized by in vitro transcription with either DIG-11-UTP (Sigma-Aldrich, MO, USA) or Fluorescein-12-UTP (Sigma-Aldrich) using either the T3 or SP6 riboprobe system (Promega, WI, USA) with modifications from the manufacturer's suggested protocol. The transcription reaction volume was reduced to 10 μl and 0.5 units of thermostable inorganic pyrophosphatase (New England Biolabs, MA, USA) was included in the reaction. Following the transcription reaction, the DNA template was degraded by treatment with DNase (Promega, WI, USA) before being analyzed on a 1% agarose gel. Successful reactions were diluted 1:100 in prehybridization solution and stored at -20°C.

Gaetano A.J, & King R.S. (2023). A simplified and rapid in situ hybridization protocol for planarians. BioTechniques, 75(6).

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