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Dig labeled ddutp

Manufactured by Roche
Sourced in United Kingdom

Dig labeled ddUTP is a nucleotide analog used in DNA sequencing and related molecular biology applications. It serves as a terminator in the chain-termination method of DNA sequencing, allowing for the detection and analysis of DNA sequences.

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2 protocols using dig labeled ddutp

1

Biochemical Toolkit for DNA Manipulation

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Restriction enzymes, vectors (pTWIN1 & pMBX10), DNA (ØX virion, ØX dsDNA and M13mp18 dsDNA), E. coli Topoisomerase I and chitin beads were obtained from New England Biolabs, UK. Q-Sepharose, Sephadex 50 columns, terminal transferase, Dig labeled ddUTP, anti-Dig antibody and NBT-BCIP solution were obtained from Roche Life Sciences, UK. Ni–NTA matrix was obtained from Qiagen, Germany. IPTG, ATP, phosphocreatine, phosphocreatine kinase and E. coli Ssb protein were obtained from Sigma–Aldrich India. Bacterial growth medium component were obtained from BD and Co., India. Oligo dT50 was obtained from MWG Biotech, India. Genomic DNA isolation kit was obtained from Hi-media Laboratories, India.
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2

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′.
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