Dig ddutp

Manufactured by Roche

Sourced in United States

DIG-ddUTP is a nucleotide analog used in various molecular biology techniques. It contains a digoxigenin (DIG) label, which can be detected using anti-DIG antibodies. DIG-ddUTP can be incorporated into DNA or RNA during enzymatic synthesis, allowing for the labeling of nucleic acid molecules.

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Lab products found in correlation

Binding buffer, by Roche (3 mentions) Terminal transferase, by Roche (2 mentions) Ecl film, by GE Healthcare (1 mentions) Ecl film, by Cytiva (1 mentions) Rnaiso plus, by Takara Bio (1 mentions) Dig labeled ddutp, by Roche (1 mentions)

Close spelling variants of this product

Dig labeled ddUTP DIG)-11-ddUTP

6 protocols using dig ddutp

Investigating CRP Binding in Shewanella lipBA Operon

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The function of the predicted CRP-binding site of Shewanella lipBA operon was assessed in vitro using electrophoretic mobility shift assays (EMSA) with little improvements (Feng and Cronan 2011a (link); Goble et al. 2013 (link); Feng et al. 2014 (link)). In the EMSA tests, nine pieces of DNA probes were composed of seven suspected probes (lipBA_she, ybeD_ec, ybeD_es, ybeD_kp, ybeD_st1, ybeD_st2, and ybeD_yp) and the two control probes, the fadD_ec site with known function (the positive control) and the lipA_ec without any function (the negative control) (Table3). The digoxigenin (DIG)-labeled DNA probes were prepared in vitro through annealing two complementary oligonucleotides in TEN buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, 100 mmol/L NaCl; pH 8.0) and then labeled by the terminal transferase with DIG-ddUTP (Roche, Indianapolis, IN, USA) (Feng et al. 2014 (link)).
In the presence/absence of cAMP (20 pmol), the various DIG-labeled DNA probes (0.2 pmol) were incubated with or without CRP protein in the binding buffer (Roche) at room temperature for around 20 min. Following the separation of the DNA-protein complexes with a native 7% PAGE gel, the chemiluminescent signals were further captured by the exposure to ECL film (GE Healthcare, Piscataway, NJ, USA) (Feng and Cronan 2011b (link), 2012 (link)).

Zhang H., Luo Q., Gao H, & Feng Y. (2015). A new regulatory mechanism for bacterial lipoic acid synthesis. MicrobiologyOpen, 4(2), 282-300.

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EMSA Assay for PdhR Binding

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The binding abilities of the putative PdhR-binding palindromes were evaluated using EMSA tests with minor modifications [20 (link), 17 (link), 14 (link)]. The digoxigenin (DIG)-labeled DNA probes were prepared in vitro through annealing two complementary oligonucleotides (Table 2) suspended in TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl; pH 8.0) followed by the terminal transferase-aided labeling with DIG-ddUTP (Roche). The DNA probes were lipA1, lipA2 (which provided a negative control), lipA3, lipA3D (the constructed palindromic version of the lipA3 site) and pdhR (which provided a positive control) (Table 2). After 16 min of incubation of the DIG-labeled DNA probes (0.2 pmol) with or without PdhR in binding buffer (Roche) at room temperature, the DNA-protein complexes were separated using a native 7% PAGE gel, and the chemiluminescent signal captured by exposure to ECL film (Amersham) [15 (link), 16 (link)].

Feng Y, & Cronan J.E. (2014). PdhR, the Pyruvate Dehydrogenase Repressor, Does not Regulate Lipoic Acid Synthesis. Research in microbiology, 165(6), 429-438.

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FadR-DNA Interaction Assay Protocol

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Electrophoretic mobility shift assays were performed to address interaction between FadR and series of taget DNA probes (e.g., fadD and fadL (Feng & Cronan, 2012 (link))) essentially as previously reported (Feng & Cronan, 2011b (link), Feng & Cronan, 2011a (link), Feng & Cronan, 2009b (link)). With an exception of fadM probe (PCR products) (Feng & Cronan, 2009b (link)), all of the FadR-recognizable probes were prepared by annealing two complementary primers (Table 2) by incubation in TEN buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, 100 mmol/L NaCl, pH 8.0) at 95°C for 5 min followed by slow cooling to 25°C and then digoxigenin (DIG) labeling by terminal transferase with DIG-ddUTP (Roche). The DIG-labeled DNA probes (either 0.1 pmol or 0.2 pmol) were incubated with either DNA binding protein in binding buffer (Roche) for 15 min at room temperature and then analyzed by native PAGE (7–7.5% PAGE for all probes).

Zhang Y., Gao R., Ye H., Wang Q, & Feng Y. (2014). A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein. Protein & Cell, 5(12), 928-939.

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High-Salt Nuclear Protein Extraction

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Nuclear extracts from TE671 were prepared using high salt lysis/extraction buffer: 10 mM NaCl, 20 mM HEPES [pH 7.6], 1.5 mM MgCl₂, 1 mM ZnSO₄, 20% glycerol, 0.1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl ﬂuoride (38 (link)). The oligonucleotides corresponding to the BSR-fragments were annealed (sense and antisense) and DIG-end-labeled with terminal transferase (Roche) and DIG-ddUTP (Roche).

Kim E., Rich J., Karoutas A., Tarlykov P., Cochet E., Malysheva D., Mamchaoui K., Ogryzko V, & Pirozhkova I. (2015). ZNF555 protein binds to transcriptional activator site of 4qA allele and ANT1: potential implication in Facioscapulohumeral dystrophy. Nucleic Acids Research, 43(17), 8227-8242.

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miRNA Detection and Quantification Protocol

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For miRNA gel blot analysis, total RNA samples were extracted from plant materials using RNAiso Plus (Takara), with a few modifications as reported previously (Jung et al. [2007 (link)]). After isopropanol precipitation, the Eppendorf tube containing the RNA pellets was briefly centrifuged without rinsing with ethanol to improve both the yield and solubility of miRNA in total RNA preparations. RNA gel blot analyses were performed with 5 μg of total RNA. Locked nucleic acid (LNA) 5′-ATgCAgCAtCAtCAaGAtTCT-3′ (upper- and lower-case letters indicate DNA and LNA, respectively) was used as an antisense oligonucleotide probe for miR172 (Varallyay et al. [2008 (link)]). The miR172 LNA probe was either labeled with P³² or 3′-end-labeled with DIG-ddUTP (Roche, Mannheim, Germany). Levels of the primary miR172 (pri-miR172) transcripts were measured by qRT-PCR using the primer pairs described in Additional file 2: Table S1.

Lee Y.S., Lee D.Y., Cho L.H, & An G. (2014). Rice miR172 induces flowering by suppressing OsIDS1 and SNB, two AP2 genes that negatively regulate expression of Ehd1 and florigens. Rice, 7, 31.

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Oligonucleotide 3'-end Labeling with DIG-ddUTP

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terminal transferase was used to label oligonucleotides at their 3′- ends by incorporation of a single DIG-labeled ddUTP (Roche Diagnostics, Mannheim, Germany). Briefly, 200 pmol oligonucleotides were incubated with 1 μL of 1 mM DIG-ddUTP and 20 U of terminal transferase (Roche) in 1 × reaction buffer and 5 mM CoCl₂ to a final volume of 20 μL at 37 °C for 60 min, and the reaction was stopped by adding 2 μL 0.2 M EDTA, pH 8.0. For the preparation of 18S rRNA probes, 18S rDNA was labeled by random priming with DIG-dUTP using DIG-High Prime kits (Roche). One microgram of 18S rDNA, amplified by polymerase chain reaction (PCR) and purified by agarose gel extraction, was mixed with the 5 × random primer mix supplied, and incubated at 37 °C overnight. The primers used for 18S rDNA amplification by PCR were 5′–AAC CTC GGG CCG ATC GCA CG–3′ and 5′–TCA AAG TAA ACG CTT CGG GC–3′.

Oh B.K., Choi Y., Bae J., Lee W.M., Hoh J.K, & Choi J.S. (2019). Increased amounts and stability of telomeric repeat-containing RNA (TERRA) following DNA damage induced by etoposide. PLoS ONE, 14(11), e0225302.

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