To determine the differences of mt tRNALeu(UUR) structures, a melt curve analysis was performed by using 1:10 000 SYBR Green I Nucleic Acid Gel Stain (Thermo Fisher Scientific, S7563) with reference to the previous method (34 (link)). First, a 10 × SYBR Green I Buffer was prepared in 100 mM Tris–HCl, pH 7.5, 20 mM MgCl2, 1.5% (v/v) Triton X-100 and 1.5 M NaCl by diluting the original dye 1000-fold. To prepare samples for the melt curve analysis, purified tRNA samples were incubated at 65°C for 5 min and then at room temperature for 45 min. For RNase A treatment of mt tRNALeu(UUR), wild-type tRNALeu(UUR) was incubated with 2 μg/μl RNase A (Wako, 182-01493) at 37°C for 5 min. Then, tRNA samples (wild-type tRNALeu(UUR), tRNALeu(UUR) with 3243A > G mutation, tRNALeu(UUR) with 3243A > G mutation and 3290T > C SNP, RNase A-treated wild-type tRNALeu(UUR), and no RNA) were diluted in the 1 × SYBR Green I Buffer. The melt curve analysis was conducted using a 20 μl sample volume on the StepOnePlus Real-Time PCR System (Applied Biosystems) in triplicate with a temperature gradient of 0.5°C /s from 50 to 95°C.
Rnase a
Manufactured by Fujifilm
Sourced in Japan
RNase A is a laboratory product from Fujifilm. It is an enzyme that cleaves single-stranded RNA, making it a useful tool for various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Achromopeptidase, by Fujifilm (6 mentions)
Lysozyme, by Merck Group (6 mentions)
Proteinase k, by Merck Group (3 mentions)
Propidium iodide, by Fujifilm (3 mentions)
Phenol chloroform isoamyl alcohol, by Thermo Fisher Scientific (2 mentions)
Epics altra, by Beckman Coulter (2 mentions)
Proteinase k, by Fujifilm (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green 1 nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Lysozyme, by Fujifilm (1 mentions)
Purified achromopeptidase, by Fujifilm (1 mentions)
Sodium dodecyl sulfate (sds), by Fujifilm (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
7 aad, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Annexin 5 apc and 7 aad, by BD (1 mentions)
Fv1200, by Olympus (1 mentions)
Hexadecyltrimethylammonium bromide, by Fujifilm (1 mentions)
Bovine pancreatic trypsin, by Fujifilm (1 mentions)
27 protocols using rnase a
1
Melt Curve Analysis of mt tRNA Leu(UUR) Structures
2
Bacterial DNA Extraction from Mouse Stool
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Stool samples collected from mice were immediately frozen using liquid nitrogen and stored at −80 °C until use. Bacterial genomic DNA was isolated as described previously with modifications51 (link)52 (link). The bacterial pellet was suspended and incubated with lysozyme (15 mg/ml, Wako Pure Chemical Industries, Ltd) at 37 °C for 1 h in 100 mM Tris-HCl/10 mM EDTA (10 × TE). Purified achromopeptidase (Wako Pure Chemical Industries, Ltd) was added at a final concentration of 2000 U/ml and then incubated at 37 °C for 30 min. The suspension was treated with 1% (wt/vol) sodium dodecyl sulfate (Wako Pure Chemical Industries, Ltd) and proteinase K (1 mg/ml, Merck Japan, Tokyo, Japan) and incubated at 55 °C for 1 h. The lysate was treated with phenol/chloroform/isoamyl alcohol, and DNA in the aqueous phase was precipitated by the addition of ethanol and pelleted via centrifugation at 5,000 × g at 4 °C for 15 min. The DNA pellet was rinsed with 75% ethanol, dried, and dissolved in 1 × TE. DNA samples were purified by treatment with RNase A (1 mg/ml, Wako Pure Chemical Industries, Ltd) at 37 °C for 30 min and precipitated by the addition of equal volumes of a 10% polyethylene glycol solution (PEG6000-2.5 M NaCl). DNA was pelleted via centrifugation at 18,000 × g at 4 °C for 10 min, rinsed with 75% ethanol, and dissolved in 1 × TE.
3
Extraction and Purification of Bacterial DNA
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Fecal samples were collected from Ccr9+/+ and Ccr9−/− mice and immediately frozen at −80 °C. The bacterial pellet was suspended and incubated with lysozyme (Sigma-Aldrich) at 37 °C for 1 h in TE10 (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Achromopeptidase (Wako Pure Chemical) was added, and samples were incubated at 37 °C for 30 min. The suspension was treated with 20% sodium dodecyl sulfate and proteinase K (Merck, Rahway, NJ, USA), incubated at 55 °C for 1 h, and treated with phenol/chloroform/isoamyl alcohol (Invitrogen). The DNA pellet was rinsed with 75% ethanol and dried. DNA samples were purified by RNase A (Wako) treatment and precipitated with 20% polyethylene glycol solution (20% PEG-2.5 M NaCl). DNA was then pelleted by centrifugation, rinsed with 75% ethanol, and dissolved in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) buffer.
4
Cell Cycle and Apoptosis Analysis by Flow Cytometry
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The percentage of cells in different phases of the cell cycle, including the sub-G1 population, was measured by flow cytometry using propidium iodide (PI, Wako) staining46 . In brief, cells were treated with each compound for the indicated time, fixed with 70% ethanol at 4°C, and then stained with 50 μg/mL PI and 10 μg/mL RNase A (Wako) for 20 min at 37°C. PI fluorescence was measured on an EPICS ALTRA (Beckman Coulter, Brea, CA).
Detection of early apoptotic cells was determined by using annexin V/APC and 7AAD (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Briefly, 1 × 105 cells were exposed to each compound and washed with PBS twice. They were then incubated at room temperature with annexin V/APC and 7-AAD for 15 min. Annexin V/APC- and 7-AAD-stained cells were enumerated with a FACSCanto (BD Biosciences). Annexin V/APC-positive or -negative cells were regarded as apoptotic and non-apoptotic cells, respectively.
In the vector-transfection experiments, we co-transfected the EGFP expression vector as a transfectant marker with DN-TCF7L2 (Merck Millipore, Billerica, MA) or an active, mutated human β-catenin (S37A and S45A) expression vector (Daiichi Sankyo Co. Ltd.). EGFP-negative group were gated out by the cutoff results of non-transfectant during FCM analysis.
Detection of early apoptotic cells was determined by using annexin V/APC and 7AAD (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Briefly, 1 × 105 cells were exposed to each compound and washed with PBS twice. They were then incubated at room temperature with annexin V/APC and 7-AAD for 15 min. Annexin V/APC- and 7-AAD-stained cells were enumerated with a FACSCanto (BD Biosciences). Annexin V/APC-positive or -negative cells were regarded as apoptotic and non-apoptotic cells, respectively.
In the vector-transfection experiments, we co-transfected the EGFP expression vector as a transfectant marker with DN-TCF7L2 (Merck Millipore, Billerica, MA) or an active, mutated human β-catenin (S37A and S45A) expression vector (Daiichi Sankyo Co. Ltd.). EGFP-negative group were gated out by the cutoff results of non-transfectant during FCM analysis.
5
Immunofluorescence Analysis of Autophagy
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Infected cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 solution in PBS for 10 min, followed by incubation in 3% bovine serum albumin/PBS + 10 mg/ml RNaseA (Wako Pure Chemical Industries Ltd., Osaka, Japan) overnight at 4°C. The cells were incubated with primary antibodies for 60 min and secondary antibodies for 30 min, followed by three 5 min-washes in PBS. Finally, cells were stained with propidium iodide nucleic acid stain (0.5 μg/ml, Invitrogen-Molecular Probes) for 40 min and mounted with Fluor SaveTM Reagent (Calbiochem, Billerica, MA, USA). All steps were performed at room temperature. The cells were observed under a fluorescence laser-scanning microscope (FV1200, Olympus, Tokyo, Japan). The degree of autophagy induction was determined by the frequency (%) of cells with at least one LC3-positive vacuole containing P. acnes in 100 to 150 randomly-counted P. acnes-infected cells.
6
DNA Extraction and Purification Protocol
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We extracted and purified DNA from the samples according to a literature with minor modifications [28 ].
In brief, after thawing, we filtered the samples using 100 μm mesh and washed them with PBS. The bacterial pellets were treated with lysozyme (Sigma-Aldrich Japan, Tokyo, Japan). Then the samples were treated with achromopeptidase (Wako Pure Chemical Industries).
The DNA was purified by SDS (Wako Pure Chemical Industries)/ proteinase K (Merck Japan, Tokyo, Japan) treatment, followed by phenol/chloroform extraction. After incubation with RNase A (Wako Pure Chemical Industries), sample DNA was precipitated with polyethylene glycol solution (Wako Pure Chemical Industries).
The samples were assessed by measuring the ratio of optical density at 260 nm to that at 280 nm (typically 1.66 to 2.1). We then confirmed the amplicon libraries using agarose gel electrophoresis.
In brief, after thawing, we filtered the samples using 100 μm mesh and washed them with PBS. The bacterial pellets were treated with lysozyme (Sigma-Aldrich Japan, Tokyo, Japan). Then the samples were treated with achromopeptidase (Wako Pure Chemical Industries).
The DNA was purified by SDS (Wako Pure Chemical Industries)/ proteinase K (Merck Japan, Tokyo, Japan) treatment, followed by phenol/chloroform extraction. After incubation with RNase A (Wako Pure Chemical Industries), sample DNA was precipitated with polyethylene glycol solution (Wako Pure Chemical Industries).
The samples were assessed by measuring the ratio of optical density at 260 nm to that at 280 nm (typically 1.66 to 2.1). We then confirmed the amplicon libraries using agarose gel electrophoresis.
7
DNA Extraction from Seaweed Samples
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Samples (G. chorda) were collected on the coast of Hakodate, Hokkaido Prefecture, Japan. Hexadecyltrimethylammonium Bromide (CTAB), tris-[hydroxymethyl]amino-methane (Tris), ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium salt, dihydrate (EDTA), Proteinase K (EC 3.4.21.64, from Tritirachium album), RNase A (EC 3.1.27.5, from bovine pancreas), porcine stomach pepsin (EC 3.4.23.1) and bovine pancreatic trypsin (EC 3.1.21.4) were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Phenol-chloroform-isoamyl alcohol (25:24:1) was purchased from NACALAI TESQUE, INC (Kyoto, Japan). Ala-Pro-p-nitroanilide (Ala-Pro-pNA) were also obtained from Bachem AG (Bubendorf, Switzerland). All other regents were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan).
8
Antibody Validation and Reagent Sourcing
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The following antibodies were purchased from suppliers: Bcl-xL (#sc-634), CHOP (#sc-793), DDX5 (#sc-365164), DDX17 (#sc-398168), DDX21 (#sc-376758), GST (#sc-138), and JNK1 (#sc-571), Santa Cruz Biotechnology (Dallas, TX, USA); Bim (#2933), Cleaved Caspase-3 (#9661), Fibrillarin (#2639), GAPDH (#2118), and phospho-JNK (#9255), Cell Signaling Technology (Danvers, MA, USA); FLAG (#F1804) and horseradish peroxidase (HRP)-conjugated FLAG (#A8592), Sigma-Aldrich (St. Louis, MO, USA); TDP-43 (#12892-1-AP), Proteintech Group (Rosemont, IL, USA); HA (#11867423001) and HRP-conjugated HA (#12013819001), Roche Diagnostics (Basel, Swiss); DDX18 (#A300-636A), Bethyl Laboratories (Montgomery, TX, USA); Myc (#BML-SA294-0500), Enzo Life Sciences (Farmingdale, NY, USA). AS601245 and CX5461 were purchased from Merck Millipore (Burlington, MA, USA) and AdooQ BioScience (Irvine, CA, USA), respectively. RNase A were purchased from Wako (Osaka, Japan).
9
Virus component enzymatic treatment
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After ultracentrifugation at 450,000 g for 30 min at 4°C, virus components (equivalent to two female) were treated by 0.01 μg/μl trypsin (Sigma-Aldrich, USA) at 25°C for 12 h, 0.2 units/μl DNase I (Takara, Japan) at 37°C for 2 h, 0.2 units/μl Benzonase (Merck, USA) at 37°C for 2 h, or 0.1 units/μl RNase A (Wako, Japan) at 37°C for 2 h. After each reaction, the reaction mixture was diluted 32 times and injected into test Drosophila larvae. Each ernzyme reaction mixture without virus components was used as a control sample.
10
Streptozotocin-based Diabetic Model
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Streptozotocin (STZ), urea, thiourea, sodium dodecyl sulfate (SDS), 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), bromophenol blue, iodoacetamide, RNase A, and DNase I were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Source information for all other assay reagents and materials is stated in the Materials and Methods section described below.
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