Digoxigenin labelled nucleotides

Manufactured by Roche

Digoxigenin labelled nucleotides are synthetic DNA or RNA molecules that have been modified by the addition of a digoxigenin label. Digoxigenin is a steroid compound that can be used to detect and locate specific DNA or RNA sequences in various applications, such as in situ hybridization, Southern blotting, and Northern blotting. These labelled nucleotides can be incorporated into the target sequence during amplification or synthesis, allowing for the subsequent detection and analysis of the labelled molecules.

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Lab products found in correlation

Anti digoxigenin ap antibody, by Roche (1 mentions) Rnaeasy kit, by Qiagen (1 mentions) Nbt bcip chromogenic substrate, by Roche (1 mentions) Apoptag kit, by Merck Group (1 mentions) Rna polymerase, by Promega (1 mentions) Igg2b, by Merck Group (1 mentions) Z vad fmk, by Merck Group (1 mentions) Cryostat, by Leica camera (1 mentions) γ tubulin, by Merck Group (1 mentions) Bromodeoxyuridine (brdu), by Roche (1 mentions) T7 rna polymerase, by Roche (1 mentions)

3 protocols using digoxigenin labelled nucleotides

In Situ Hybridization Probe Synthesis

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To prepare in situ hybridization probes, DNA templates were obtained by linearization of plasmids containing atoh7 and ccnd1 cDNAs using the EcoRI and NotI restriction enzymes, respectively. RNA probes were synthesised by in vitro transcription. Appropriate polymerases (T7, T3; Promega) and digoxigenin-labelled nucleotides (Roche) were used for the synthesis of antisense RNAs according to manufacturer's instructions. Synthesized probes were purified using RNAeasy kit (Qiagen). Embryos were fixed and processed as previously described (Thisse and Thisse, 2008) and hybridization signals were detected using anti-digoxigenin-AP antibody (1:4000; 11093274910, Roche) and the NBT/BCIP chromogenic substrate (1:3.5; Roche). After the procedure, embryos were fixed and stored at 4°C until imaging

Wycliffe R., Plaisancie J., Leaman S., Santis O., Tucker L., Cavieres D., Fernandez M., Weiss-Garrido C., Sobarzo C., Gestri G, & Valdivia L.E. (2021). Developmental delay during eye morphogenesis underlies optic cup and neurogenesis defects in mab21l2^u517 zebrafish mutants. The International journal of developmental biology, 65(4-5-6).

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Immunolabelling and In Situ Hybridization in Zebrafish

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Whole-mount immunolabelling procedures were performed as previously described [34 (link),76 (link)]. For antibody staining of cryosections, embryos were first protected by sequential incubation in 15% then 30% sucrose in phosphate-buffered saline supplemented with 0.5% Triton X-100 (PBST) for 12–16 hours at 4°C, embedded in OCT, stored at –80°C, and sectioned at 16–20μm using a Leica cryostat. γ-tubulin (Sigma; 1:200); GFP (AMS Biotechnology TP401; 1:1000); anti-acetylated tubulin (IgG2b, Sigma; 1:500); anti-SV2 (IgG1, DSHB; 1:500); BrdU (Roche; 1:300); PH3 (Upstate Biochemical; 1:500);Rho4D2(Abcam; 1:200); zpr-1 (ZIRC; 1:50) antibodies were used.
Antisense mRNA probes for whole-mount in situ hybridisation were synthesised using RNA polymerases (Promega) and digoxigenin labelled nucleotides (Roche) following manufacturer's instructions. Whole-mount in situ hybridisations were performed essentially as previously described [33 (link)]. TUNEL labelling to detect apoptosis was performed using the ApopTag Kit (Chemicon International). In ordered to block apoptosis, 24hpf embryos were treated with 300 μM of caspase inhibitor (Z-VAD-FMK, Sigma).

Turner K.J., Hoyle J., Valdivia L.E., Cerveny K.L., Hart W., Mangoli M., Geisler R., Rees M., Houart C., Poole R.J., Wilson S.W, & Gestri G. (2019). Abrogation of Stem Loop Binding Protein (Slbp) function leads to a failure of cells to transition from proliferation to differentiation, retinal coloboma and midline axon guidance deficits. PLoS ONE, 14(1), e0211073.

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Synthesis of Digoxigenin-Labeled RNA Probes

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RNA antisense probes were synthesized by transcription of linearized DNA from plasmids or from cDNA amplified with specific primers (S2 Table ). Transcription was carried out with digoxigenin labelled nucleotides (Roche) and T7 RNA polymerase (Roche). Synthesized RNA was precipitated with 0.8M ammonium acetate in 75% ethanol or 0.1M LiCl in 75% ethanol and finally resuspended in 50% formamide-50%
RNase free water.

López-Delgado A.C., Delgado I., Cadenas V., Sánchez‐Cabo F., & Torres M. (2020). Axial skeleton anterior-posterior patterning is regulated through feedback regulation between Meis transcription factors and retinoic acid.

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