The single-stranded DNA (ssDNA)-specific S1 endonuclease from Aspergillus oryzae (Invitrogen, Life Technologies) (35 (link)) was used to generate double-strand breaks (DSB) from UV-induced ssDNA sites (36 (link)). Lesions were resolved in a single gel electrophoresis assay (comet assay) as previously described (24 (link)) with modifications for the use of S1 nuclease. The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C. As a control, the other half of each slide was incubated with the same solution, but without the nuclease. See Supplementary Material for more details. Comets were stained with ethidium bromide, imaged with a fluorescence microscope (Olympus BX51, Olympus, Center Valley, PA, USA), and at least 50 comets were scored for each condition per slide with Kinetic Imaging Komet 6.0 (Andor TM Technology, Belfast, UK). Independent experiments were performed four times in duplicate.
S1 nuclease
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy, United Kingdom, China
S1 nuclease is an enzyme that cleaves single-stranded DNA and RNA. It specifically digests the phosphodiester bonds in single-stranded nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (21 mentions)
Dnase 1 amplification grade, by Thermo Fisher Scientific (5 mentions)
Rnase v1, by Thermo Fisher Scientific (5 mentions)
Proteinase k, by Thermo Fisher Scientific (5 mentions)
Chef dr 3 system, by Bio-Rad (4 mentions)
Trizol plus rna purification kit, by Thermo Fisher Scientific (3 mentions)
Rna clean concentrator 5, by Zymo Research (3 mentions)
Chef dr 2 system, by Bio-Rad (3 mentions)
Rnase t1, by Thermo Fisher Scientific (3 mentions)
Alkaline phosphatase, by Thermo Fisher Scientific (3 mentions)
Ethidium bromide, by Merck Group (3 mentions)
Rnase a, by Merck Group (3 mentions)
Photoprobe biotin, by Vector Laboratories (3 mentions)
Xbai, by Thermo Fisher Scientific (3 mentions)
Fastprep 24, by MP Biomedicals (2 mentions)
B02 compound, by Selleck Chemicals (2 mentions)
Smarter race 5 3 kit, by Takara Bio (2 mentions)
Chef dr 3, by Bio-Rad (2 mentions)
5 ethynyl 2 deoxyuridine (edu), by Merck Group (2 mentions)
Zymoclean gel rna recovery kit, by Zymo Research (2 mentions)
139 protocols using s1 nuclease
1
Quantifying DNA Damage Using S1 Nuclease
2
DNA Fiber Assay for Replication Stress
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Cells were incubated with 100μM IdU and 100μM CldU as indicated. Chemical compounds (HU, cisplatin, olaparib) were added according to the labeling schemes presented. Next, cells were collected and processed using the FiberPrep kit (Genomic Vision EXT-001) according to the manufacturer’s instructions. Samples were added to combing reservoirs containing MES solution (2-(N-morpholino) ethanesulfonic acid) and DNA molecules were stretched onto coverslips (Genomic Vision COV-002-RUO) using the FiberComb Molecular Combing instrument (Genomic Vision MCS-001). For S1 nuclease assays, MES solution was supplemented with 1mM zinc acetate and either 40U/mL S1 nuclease (ThermoFisher 18001016) or S1 nuclease dilution buffer as control, and incubated for 30 minutes at room temperature. Slides were then stained with antibodies detecting CldU (Abcam 6236) and IdU (BD 347580), and incubated with secondary AF488 (Abcam 150117) or Cy5 (Abcam 6565) conjugated antibodies. Finally, the cells were mounted onto coverslips and imaged using a confocal microscope (Leica SP5) and analyzed using LASX 3.5.7.23225 software.
3
Genomic DNA Extraction and Nucleoside Analysis
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The genomic DNA of gonads were extracted using DNAiso Reagent (Takara Biotechnology, Dalian, China) according to the manufacture’s recommended procedure. The concentration of the genomic DNA was determined by a BioPhotometer (RS232C, Eppendorf, Hamburg, Germany). Genomic DNA was digested by S1 Nuclease (EN0321, Thermo Scientific, Rockford, IL, USA) at 37 °C with water bath for 16 h, and the total volume of reaction system was 10 µl with 10 µg genomic DNA, 1 µl 10×S1 Nuclease buffer, 90 U S1 Nuclease. Then, 9 U alkaline phosphatase (18009027, Invitrogen, Carlsbad, USA) and 0.002 U phosphodiesterase I (P4506, Sigma-Aldrich, St Louis, USA) were added to the reaction system of 40 µl simultaneously. The reaction was incubated in water bath at 37 °C for 4 h. After enzyme digestion, the samples were extracted by phenol and chloroform. The nucleoside samples were analyzed by LC–ESI–MS/MS.
4
DNA Fiber Assay for Replication Forks
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The progression of replication forks upon UV exposure was evaluated by a DNA fiber assay, with a 20-min pulse of chlorodeoxyuridine (CldU, Sigma-Aldrich) before UVC irradiation and a 60-min pulse of iododeoxyuridine (IdU) afterward (24 (link)). For experiments with cells expressing photolyases, upon UV irradiation, the cells were incubated with 200 μM IdU in PBS+ supplemented with 5% FBS for 60 min at RT on the photoreactivation apparatus. For experiments with the ssDNA-specific S1 endonuclease, after an IdU pulse, the cells were treated with CSK100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM MOPS, 0.5% Triton X-100) for 10 min at RT, then incubated with S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 10 mM zinc acetate and 5% glycerol) with or without S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml for 30 min at 37ºC. See Supplementary Material for more details. DNA fibers were imaged using a fluorescent microscope (Axiovert 200, Zeiss, Jena, Germany) at a magnification of 1000×. Analyses were performed using Zeiss LSM Browser Software. All experiments were performed at least twice independently, and at least 100 fibers were counted for each slide. Results repeated in separate figures represent independent experiments.
5
Detecting ssDNA Gaps in Nascent DNA
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Detection of ssDNA gaps on nascent DNA was performed as already described (45 (link)) with minor modifications. Analogue pulses and treatments were performed as described above and in the figure legends. Cells were then extracted in 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl2, 10 mM PIPES pH 6.8, 0.5% Triton X-100 for 7 min at room temperature and incubated for 20 min at 37°C in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 2 mM zinc acetate and 5% glycerol) supplemented or not with 20 U ml–1 of S1 nuclease (Invitrogen). Nuclei were collected by scrapping in PBS 0.1% BSA followed by centrifugation at 1500 × g for 5 min at 4°C. Nuclei were resuspended in PBS 10 mM EDTA. DNA was spread and immune-stained as described above. Images were acquired on an Axio Observer Z1 microscope using Axio Vision software (Zeiss) and analysed on ImageJ software. All bicolor replication signals were included in the analysis.
6
ToxIPa Antisense Probe Generation
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An antisense probe covering the complete ToxIPa sequence was made by amplification of the ToxIPa locus from plasmid pTA110, using primers PF217 and PF218, and subsequent in vitro transcription and gel extraction of the probe as described previously (55 (link)), generating a uniformly [32P]UTP-labeled antisense transcript. Ten micrograms of DNase-treated total RNA was hybridized to the antisense probe overnight at 68°C in a total volume of 30 μl containing 22% or 6% formamide for the ΦM1 or ΦM1-O total RNA, respectively, 40 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)]-KOH (pH 6.4), 1 mM EDTA, and 400 mM NaCl. Reaction mixtures were treated with S1 nuclease (Invitrogen) (1 U μl−1) for 1.5 h at 37°C in a total volume of 300 μl 1× S1 nuclease buffer to degrade any single-stranded nucleic acids. Double-stranded hybridization products were precipitated, resuspended, and resolved by 10% PAGE. Bands were visualized by phosphorimaging (Bio-Rad Personal FX phosphorimager).
7
Triplex Formation Detection in Minicircle DNA
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To determine whether triplex formation between TFO1R and minicircle DNA had occurred, samples were probed with S1 nuclease. To prepare the triplex complex, an excess of TFO1R (2.5 µM) was incubated with the minicircle/plasmid (150 nM) in 100 mM calcium acetate pH 4.8, in a total volume of 20 µL at room temperature for 30 min. (In control experiments, reactions were also carried out in TF buffer: 50 mM sodium acetate pH 5.0, 50 mM NaCl, 50 mM MgCl2.) Aliquots (5 µL) were taken and S1 nuclease (0–1000 U; Thermo Fisher Scientific) was then added and the incubation continued in S1 nuclease buffer (30 mM sodium acetate pH 4.6, 1 mM zinc acetate, 50% [v/v] glycerol) at room temperature for 30 min; the total volume of these reactions was 10 µL. The digest was stopped by the addition of 0.25 M EDTA (5 µL), followed by heat inactivation at 70 °C for 10 min; DNA was isolated by extraction with chloroform:isoamyl alcohol.
8
Detection of ssDNA Gaps Post-Replication
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Detection of post-replicative ssDNA gaps was carried out as previously described43 (link). In brief KB1P-G3 cells, were pulse labelled with 25 μM CldU (15’) followed by 250 μM IdU (45’) with or without 50 nM CPT. Cells were then harvested by trypsinization, washed with PBS and pre-extracted with CSK buffer (100 mM NaCl, 10 mM MOPS pH 7, 3 mM MgCl2, 300 mM sucrose and 0.5% Triton X-100 in water) for 10’ at RT. Next, isolated nuclei were harvested by centrifugation and incubated for 30’at 37 °C with S1 nuclease buffer containing or not 20 U/ml of S1 nuclease (#EN0321, Thermo Fisher Scientific). Nuclei were then pelleted, resuspended in PBS and spread onto microscope slides as previously described.
9
S1 Nuclease Digestion and TRF Analysis
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Samples were collected and prepared as described above for TRF analyses. For S1 nuclease digested samples, 20 µg of RsaI/HinfI digested DNA was digested with 100 U of S1 nuclease (Thermo Scientific #EN0321) for 45 min at room temperature. For each sample, 8 to 12 μg of digested genomic DNA was resolved overnight on a 0.5% agarose 1x TBE gel. Sample lanes were cut out, re-cast, and resolved on a 1.2% agarose 1x TBE gel. Gels were treated, transferred to Hybond-XL membrane, labeled, and imaged as described above for TRF analyses.
10
Replication Combing Assay for DNA Fibers
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