Synthetic oligoribonucleotides were obtained from GE Healthcare Dharmacon (U30, Het30) or TriLink Biotechnologies, Inc. (ppp-Het30), dT30 was obtained from IDT, Inc. ss-dsRNAs were prepared by in vitro transcription as previously described22 (link). For RNAs containing 4-thiouridine, UTP was replaced with 4-Thio-UTP (TriLink Biotechnologies, Inc.) in the transcription reactions. All RNAs were purified by denaturing PAGE followed by electroelution in an Elutrap device (Schleicher and Schuell).
4 thio utp
Manufactured by TriLink
4-Thio-UTP is a modified nucleotide used in various biochemical and molecular biology applications. It serves as a substrate for enzymatic reactions and can be incorporated into nucleic acids, such as RNA. The sulfur atom at the 4-position of the uracil base is the key structural feature of this compound.
Automatically generated - may contain errors
3 protocols using 4 thio utp
1
Synthesis and Purification of RNA Oligonucleotides
2
Photo-crosslinking of Transcribed Plasmid
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Photo-crosslinking of transcribed plasmid was carried out as described (14 (link)) with modifications. Briefly, after transcription with 4-thio-UTP (TriLink BioTechnologies), samples were treated with or without 0.2-U/μl RNase H (Fermentas, Thermo Scientific) at 37°C for 15 min. They were then transferred to a 24-well microtiter plate (Greiner Bio-One, Germany), placed on ice in a UVP CL-1000 Ultraviolet Cross-linker (UVP), and irradiated for 20 min with 365-nm ultraviolet (UV) light at a distance of 4–5 cm. DNA was recovered with phenol/chloroform extraction and ethanol precipitation. Next, primer extension was performed with a 5′-FAM-ATTACAGGCGAACATACTTAC-3′ primer and Thermo Sequenase from the Cy5 Dye Terminator Cycle Sequencing Kit (GE Healthcare). Guanine and thymine ladders were prepared by primer extension in the presence of ddCTP or ddATP, in a 1/100 molar ratio to dCTP or dATP, respectively.
3
Diverse Nucleotide Analogs for Labeling
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1, cy3-dUTP (Santa Cruz Biotech.); 2, Digoxigenin-11-dUTP (Sigma); 3, Biotin-16AA-dUTP (TriLink Biotech.); 4, Texas Red-5-dCTP (PerkinElmer); 5, N6 -(6-Azido)hexyl-ATP (Jena Bioscience); 6, Cyanine 3-AA-UTP (TriLink Biotech.); 7, 4-Thio-UTP (TriLink Biotech.); 8, Biotin-16-AACTP (TriLink Biotech.); 9, Ganciclovir Triphosphate (TriLink Biotech.); 10, 5-Hydroxymethyl-2’-deoxyuridine-5’-Triphosphate (TriLink Biotech.).
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