Hydrolysis of the nucleic acids was achieved by enzymatic reactions which cleave the RNA molecule to 5′-mono-nucleotide. Three different enzymes were tested: S1 Nuclease (Promega, Madison, WI 53711-5399, USA; Cat. No. E567B), S1 Nuclease (Invitrogen- Thermo Fischer Scientific, Waltham, MA 02451, USA; Cat. No. 180001-016) and RNAase ONETM Ribunclease (Promega, Madison, WI 53711-5399, USA; Cat. No. M426A). For assessing the digestion conditions, 5 µg of each 15 bases oligo (polyA, polyC, polyG and polyU, purchased by IDT—Integrated DNA Technologies; Leuven, Belgium) as well 5 µg of a control RNA purchased by Celbio were incubated in 1× working buffer with 50–160 units of enzyme at 37 °C for two hours in a final volume of 50 µL. After digestion, the samples were immediately stored at −20 °C up to HPLC analysis. The following digestion reactions of mouse liver as well as clinical samples were carried out using about 2 µg of RNA with 80 units of S1 Nuclease (Promega, Madison, WI 53711-5399, USA) in the abovementioned conditions.
S1 nuclease
Manufactured by Promega
Sourced in United States
S1 nuclease is a single-stranded DNA and RNA-specific endonuclease that cleaves phosphodiester bonds in single-stranded nucleic acids. It is commonly used in molecular biology applications to remove single-stranded regions of DNA or RNA.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Promega (6 mentions)
Brightstar plus positively charged nylon membrane, by Thermo Fisher Scientific (3 mentions)
Chef mapper system, by Bio-Rad (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Truseq dna sample preparation kit v2, by Illumina (2 mentions)
Ovation rna seq v2 kit, by Tecan (2 mentions)
Sizeselect e gels, by Thermo Fisher Scientific (2 mentions)
Chef mapper xa system, by Bio-Rad (2 mentions)
Chef mapper, by Bio-Rad (2 mentions)
Pcr dig probe synthesis kit, by Roche (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Rnase t1, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Las 3000 imaging analyzer, by Fujifilm (1 mentions)
Sybr green 2, by Lonza (1 mentions)
Dl dithiothreitol, by Merck Group (1 mentions)
Midi kit, by Qiagen (1 mentions)
Rnase 3, by New England Biolabs (1 mentions)
Rnase a, by Merck Group (1 mentions)
53 protocols using s1 nuclease
1
Enzymatic Nucleic Acid Hydrolysis
2
In Vitro RNA-Dependent RNA Polymerase Assay
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A 5-pmol template RNA (final concentration, 100 nM) and 0.8-pmol HuNV RdRp (final concentration, 16 nM) were mixed with the reaction buffer (20-mM Tris-HCl [pH 7.4], 5-mM DL-Dithiothreitol, 2-mM NTPs (ATP, UTP, GTP, CTP; Sigma Aldrich), 2-mM MnCl2, 0.8-U/μl ribonuclease inhibitor (Toyobo)) and incubated at 30 °C. The mixture was divided into two portions (20 μl each) 30 min after incubation. To one portion, 4 μl of 6× gel-loading buffer (10% glycerol, 0.02% BPB, 0.01% xylene cyanol in TBE buffer [89-mM Tris, 89-mM borate, 2-mM EDTA]) was added, and nondenaturing polyacrylamide gel electrophoresis (native PAGE) with the running buffer (25-mM Tris, 192-mM glycine, pH 8.3) was performed at a constant voltage of 300 V for 25 min (5%–20% polyacrylamide gel [ePAGEL, ATTO]). To the other portion, 7 μl of ten times diluted S1 nuclease (89 U/μl; Promega) and 3 μl of S1 ribonuclease buffer were added and incubated for 30 min at 30 °C to degrade the ssRNA and detect double-stranded RNA (dsRNA) comprising the template RNA and the newly synthesized complementary strands. After this step, the 6× gel-loading buffer was then added, and the native PAGE was performed as described above. After native PAGE, the gel was then stained with SYBR Green II (LONZA) and detected using a LAS-3000 imaging analyzer (Fuji Film).
3
Plasmid Sizing via PFGE
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Plasmid DNA was extracted from bacteria with the Qiagen Midi Kit (Qiagen). Plasmid sizing was performed using S1-nuclease (Promega) digested plasmid DNA, followed by separation by pulsed field gel electrophoresis (PFGE) using a CHEF mapper system (Bio-Rad, USA) as previously described [21 (link)].
4
Isolation and Characterization of dsRNA from Mycelia
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Mycelia were grown in Czapek-Dox CM broth as above and total RNA samples were prepared using an RNeasy mini kit (Qiagen). LiCl fractionation of dsRNA was carried out as described [32 (link)]. Isolation of RNA from purified AfuPmV-1 was performed using phenol/chloroform treatment. DNase I (Promega), RNase A (Sigma), S1 nuclease (Promega) and RNase III (New England Biolabs) treatments of purified dsRNAs were performed according to the manufacturer’s instructions.
5
Mitochondrial Visualization and Manipulation
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Cells grown on glass coverslips were permeabilized for 2 min with CSK buffer (10 mM PIPES at pH 6.8, 300 mM sucrose, 100 mM NaCl, 1.5 mM MgCl2, 0.5% Triton) on ice and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. MitoTracker (Thermo Fisher) staining was performed at 200 nM for 20 min at 37°C according to the manufacturer's instructions. Where indicated, samples were pretreated for 30 min at 37°C with 200 mg/mL RNase A (Qiagen) and with S1 nuclease (Promega) or P1 nuclease (Sigma) plus reaction buffer. Samples were blocked for 30 min with 10% FBS in PBS and incubated with the indicated primary antibodies (0.1% FBS in PBS). Unbound primary antibodies were removed by washing four times for 5 min each in PBS followed by incubation with secondary antibodies for 45 min at room temperature. Slides were then washed four times for 5 min each in PBS before mounting with VectaShield mounting medium (Vector Laboratories) containing DAPI. Images were taken using a 63× objective on a Zeiss LSM 510 laser-scanning confocal microscope. Image analysis was carried out with Fiji (ImageJ) software.
6
Carbapenem Resistance Transfer in E. coli
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The transfer of carbapenem resistance was tested using a conjugation test, E. coli 600 (rifampicin-resistant) was used as the recipient strain, NDM-1-producing E. cloacae clinical isolates were used as the donor strains. Donor and recipient cells from M–H broth cultures were mixed in a ratio of 2:1 and transconjugant clones were screened on M–H agar plates containing rifampicin (256 mg/L) and Imipenem (1 mg/L). Conjugation events occurred at 37 °C. The presence of the blaNDM-1 gene in transconjugants was determined by PCR and sequencing. Genomic DNA was digested with S1 nuclease (Promega, USA). The linearized plasmids and partially digested genomic DNA were separated through the CHEF-Mapper XA PFGE system with a switch time from 2.16 to 63.8 s for 18 h at 6 V/cm at 14 °C. Linear plasmids generated by S1-PFGE were transferred to nvlon membrane (Millipore, USA) and hybridized with a digoxigenin-labeled probe specific to blaNMD-1. Probe labeling and signal detection were carried out with DIG high primer DNA labeling and detection starter kit according to the manufacturer’s instructions (Roche Applied Sciences, Germany).
7
Extraction and Purification of dsRNA
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Mycelia used for nucleic acid extraction were propagated on 100 mL potato dextrose broth (PDB) liquid medium in an autoclaved Ziploc container (W156 × D117 × H53 mm, Asahi Kasei, Tokyo, Japan) at 25 °C for 7 days. After collecting mycelia and draining on paper towels, 2.5 g of mycelium was homogenized in liquid nitrogen. The powder was mixed with 5 mL extraction buffer (25 mM glycine, 0.1 M NaCl, 50 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 1% SDS, 0.1% 2-Mercaptoethanol) and extracted with 5 mL phenol-chloroform twice. Total nucleic acid was precipitated with 0.3 M sodium acetate and ethanol. dsRNAs were purified from total nucleic acids using CF-11 cellulose following previously described methods [29 (link)] and by incubating with S1 nuclease (Promega, Madison, WI, USA) and DNase I (Promega) at 37 °C for 30 min.
8
Plasmid-Mediated NDM-Like Gene Detection
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Plasmid size that carried blaNDM–like genes was detected by pulsed-field gel electrophoresis (PFGE) of total DNA digested with S1 nuclease (Promega, Madison, WI, United States; Barton et al., 1995 (link)). Then the DNA was transferred to a BrightStar-Plus positively charged nylon membrane (Applied Biosystems, Foster City, CA, United States) and hybridized with digoxigenin- labeled blaNDM–like probe (Paskova et al., 2018 (link)).
9
Detection of Single-Stranded DNA
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To detect single-stranded DNA (ssDNA), the cells were fixed on ice with 4% PFA for 20 min and then with 80% methanol in PBS at − 20 °C overnight. The next day, the cells were washed and treated for 4 h at 37 °C with 200 µg/mL RNase (QIAGEN) and S1 nuclease (Promega) as indicated. Then, the cells were washed and blocked with a blocking solution (PBS containing 0.5% FBS), and then incubated overnight at 4 °C with the anti-ssDNA primary antibody. The following day, the cells were incubated with the secondary antibody for 2 h and then DAPI before mounting. Images of ssDNA were blindly captured by using confocal microscopy (LSM 510 Meta and 800; Zeiss, Göttingen, Germany) with a × 100 objective.
10
Plasmid Detection in PDD Isolates
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Seventeen PDD isolates were selected for plasmid detection on the basis of the time of detection (i.e., UME vs. non UME), haemolysis phenotype and its genetic basis (Table 1 ). Plasmids were detected by using a S1 nuclease-PFGE with the following in-house protocol. PDD isolates grown on sheep blood agar plates (bioMérieux, Craponne, France) were resuspended in a 2 ml Cell Suspension Buffer (10 mM Tris-HCl; 1 mM EDTA; pH 8.0) until reaching a turbidity of 5 McFarland. Two hundreds μl of this suspension were treated with 10 μl of proteinase K (20 mg/ml) and mixed with 100 μl of a 2% melted agarose in TE buffer. After solidification, the obtained plug was incubated for 2 h at 55⋅C in 1 ml Cell Lysis Buffer (50 mM Tris-Hcl; 50 mM EDTA; 1% sarcosyl; pH 8.0) with 5 μl of proteinase K (20 mg/ml). A 3 mm slice of the plug was restricted with 5 U of S1 nuclease (Promega, Madison, USA) for 45′ at 37⋅C. Electrophoresis was performed in a CHEF-II (Bio-Rad Laboratories GmbH, Munich, Germany) at 6 V/Cm with an initial pulse of 2.2 and a final pulse of 63.8 for 22 h. Salmonella enterica subsp. enterica serotype Braenderup H9812, restricted with XbaI (Promega) for 2 h at 37⋅C was used as size marker (Hunter et al., 2005 (link)).
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