α 32p datp

Manufactured by Cytiva

Sourced in United Kingdom

[α-32P]dATP is a radioactively labeled nucleotide that contains the phosphorus-32 isotope attached to the alpha phosphate group of deoxyadenosine triphosphate. This product is commonly used in various molecular biology and biochemical applications that require the detection and quantification of nucleic acids.

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Lab products found in correlation

Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Hyperfilm mp, by Cytiva (1 mentions) Whatman 3mm paper, by Cytiva (1 mentions) Trizol method, by Thermo Fisher Scientific (1 mentions) Phosphorimager, by GE Healthcare (1 mentions) Hybond n, by Cytiva (1 mentions) Megaprime dna labeling system, by Cytiva (1 mentions) Zeta probe nylon membrane, by Bio-Rad (1 mentions) Imagequant tl software, by GE Healthcare (1 mentions) Perfecthyb hybridization buffer, by Merck Group (1 mentions) Decaprime 2 dna labeling kit, by Thermo Fisher Scientific (1 mentions) Hybond n membrane, by Cytiva (1 mentions) Klenow buffer, by New England Biolabs (1 mentions) α 32p dctp, by Cytiva (1 mentions) Klenow fragment 3 5 exo, by New England Biolabs (1 mentions)

5 protocols using α 32p datp

Radiolabeling DNA Probe for Protein-DNA Interaction Analysis

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The P_NI DNA probe (145 bp) was obtained as described previously [21 (link)], digested with EcoRI, and labeled by filling in the overhanging EcoRI-digested end with [α-³²P]dATP (6000 Ci∙mmol⁻¹; Amersham Biosciences, Amersham, UK) and the Klenow fragment of E. coli DNA polymerase as described previously [20 (link)]. The DNA probe was mixed with the purified proteins at the concentration indicated in each assay. The retardation reaction mixtures contained 20 mM Tris-HCl pH 7.5, 10% glycerol, 2 mM β-mercaptoethanol, 50 mM KCl, 0.05 nM DNA probe, 250 μg∙mL⁻¹ bovine serum albumin, and purified His₆-BzdR protein in a 9-μL final volume. The samples were fractionated by electrophoresis in 5% polyacrylamide gels buffered with 0.5× TBE (45 mM Tris borate, 1 mM EDTA). The gels were dried onto Whatman 3MM paper and exposed to Hyperfilm MP (Amersham Biosciences, Little Chalfont, UK).

Durante-Rodríguez G., Gutiérrez-del-Arroyo P., Vélez M., Díaz E, & Carmona M. (2019). Further Insights into the Architecture of the PN Promoter That Controls the Expression of the bzd Genes in Azoarcus. Genes, 10(7), 489.

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Steady-State Levels of sRNAs in B. subtilis

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The steady-state levels of the sRNAs 13, FsrA and SurA were determined by Northern-blot analyses. B. subtilis strain 168 and strain 168Δhfq were grown in CS-glucose medium at 37°C. Aliquots for RNA purification were withdrawn at the OD₆₀₀ values indicated in Fig. 5B. RNA extraction was performed using the Trizol method (Ambion). 15 µg of total RNA were denatured for 5 min at 85°C in RNA loading dye, separated on 8% polyacrylamide-8 M urea gels, and then transferred to a nylon membrane (Amersham Hybond-N) by electroblotting. The RNA was cross-linked to the membrane by exposure to UV light. Target-specific [γ-³²P]-5′-end-labeled (Amersham Pharmacia Biotech) oligonucleotides were used as indicated in Table S4 in File S1. sRNA 13 was detected using an internally radiolabelled probe synthesized by means of PCR in the presence of [α-³²P]-dATP (Amersham Pharmacia Biotech) using primers M80/N80 (Table S4 in File S1). The hybridization signals were visualized using a PhosphorImager (Molecular Dynamics). The experiments were performed in duplicate.

Hämmerle H., Amman F., Večerek B., Stülke J., Hofacker I, & Bläsi U. (2014). Impact of Hfq on the Bacillus subtilis Transcriptome. PLoS ONE, 9(6), e98661.

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Co-transcription Analysis and Northern Blotting

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For co-transcription analysis, 100 ng of total RNA was used for synthesis of first strand cDNA. Reverse transcription was performed with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) and primer specific for the lhrC5 gene. The obtained cDNA was then used as the template for PCR performed with primers specific for internal fragments of the fri and lmo0944 genes. Primers used for the co-transcription analysis are listed in Supplementary Table S1.
For northern blotting, 20 μg of total RNA in loading buffer containing 50% formamide and 20% formaldehyde was separated on a formaldehyde agarose gel and subsequently transferred to a Zeta probe nylon membrane (Bio-Rad) by capillarity blotting. For detection of RNA, the membranes were preincubated for 1 h in PerfectHyb hybridization buffer (Sigma-Aldrich) and then hybridized overnight with a specific ³²P-labeled DNA double-stranded probes. Probes were generated using [α-32P] dATP and Megaprime DNA labeling system (Amersham Biosciences), according to the manufacturer’s protocol. Primers used for preparing DNA double-stranded probes are listed in Supplementary Table S1. RNA bands were visualized by phosphor imaging using a Typhoon scanner and analyzed with ImageQuant™ TL software (GE Healthcare).

Ładziak M., Prochwicz E., Gut K., Gomza P., Jaworska K., Ścibek K., Młyńska-Witek M., Kadej-Zajączkowska K., Lillebaek E.M., Kallipolitis B.H, & Krawczyk-Balska A. (2024). Inactivation of lmo0946 (sif) induces the SOS response and MGEs mobilization and silences the general stress response and virulence program in Listeria monocytogenes. Frontiers in Microbiology, 14, 1324062.

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Genomic DNA Preparation and Southern Blot Analysis

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Total cellular DNA was prepared according to Løbner-Olesen and von Freiesleben [60 (link)]. DNA was digested with PvuI, and fragments were separated on a 0.7% agarose gel, transferred by capillary transfer to a Hybond-N⁺ membrane (Amersham Pharmacia Biotech), and probed with an approx. 1 kb NKBOR fragment, which hybridize to NKBOR. The probe was prepared by PCR amplification using primers NKBOR_Probe_FW and NKBOR_Probe_RV (S7 Table) using pNKBOR as template and labeled with [α-³²P]dATP (Amersham Pharmacia) using the Random Primer system (DECAprime II DNA Labeling Kit; Life Technologies).

Frimodt-Møller J., Charbon G., Krogfelt K.A, & Løbner-Olesen A. (2016). DNA Replication Control Is Linked to Genomic Positioning of Control Regions in Escherichia coli. PLoS Genetics, 12(9), e1006286.

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Radiolabeling and Binding Assay for Transcription Factor

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Single-stranded purified oligonucleotides were annealed in a solution containing 0.125 M TRIS-HCl pH 7,4, NaCl 0.5 M. Annealing mixtures were heated to 95° C for 5 min and slowly chilled overnight to room temperature. About 100 ng of double-stranded oligonucleotide was 32P labeled with 5 U of Klenow fragment (3’–5’ exo-) (New England Biolabs, Beverly, MA) at 37°C for 1 h in 50 µl of a reaction mixture containing: 1× Klenow buffer (New England Biolabs), dTTP 1 mM, dGTP 1mM, 30 µCi [α32P]dATP (Amersham) and 30 µCi [α32P]dCTP (Amersham). Labeling mixtures were subsequently centrifuged through a G-25 Sephadex column to remove excess of unincorporated nucleotide. Gel retardation assays were performed by mixing 0.4 ng (about 5 × 105 cpm) of probe with 1 µl of in vitro synthesized Sp-Elk protein and 5 µg of poly(dI-dC)/poly(dI-dC) duplex in a buffer containing 20 mM HEPES (pH 7.9), 0.75 mM DTT, 57.5 mM KCl, 2.5 mM MgCl2, 0.05 mM EDTA and 10% glycerol. The binding reaction was performed on ice for 15 min and then the DNA-binding complexes were resolved by gel electrophoresis on an 8% polyacrylamide gel, 1× TBE with 300 V at 4° C. After 2–3 h run, gels were dried and exposed overnight to Biomax MS film. The sequences of oligonucleotides used are: Cy313F, 5’-GAGTATCAACAGGAAGTAGGT-3’ and Cy313R, 5’-ACCTACTTCCTGTTGATACTC-3’.

Rizzo F., Coffman J.A, & Arnone M.I. (2016). An Elk transcription factor is required for Runx-dependent survival signaling in the sea urchin embryo. Developmental biology, 416(1), 173-186.

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