The detailed protocol of EMSA has been described in previous research [58 (link),59 (link)]. Briefly, approximately 300 bp promoter fragment of the candidate gene was amplified from A. caulinodans ORS571 genome and purified as a DNA probe using the PCR purification kit (Sangon Biotech, Shanghai, China). The DNA probes (50 ng) were mixed in a binding buffer containing different concentrations of RihR protein in a final volume of 20 µL. The binding buffer contained 50 mM Tris-HCl (pH 8.3), 0.25 M KCl, 2.5 mM dithiothreitol (DTT), 5 mM MgCl2, 0.25 mg·mL−1 bovine serum albumin, 0.05 mg·mL−1 poly(dI-dC), 2.5 mM EDTA, 1% glycerol. The reaction mixtures were incubated for 20 min at room temperature and then electrophoresed on a 6% nondenaturing polyacrylamide gel in 0.5× Tris-borate-EDTA buffer at 150 V for 70 min. Gels were stained and photographed using GelRed (Sangon Biotech, Shanghai, China) and the molecular imager Gel Doc XR system (Bio Rad, Hercules, CA, USA).
Gelred
Manufactured by Sangon
Sourced in China
GelRed is a nucleic acid stain used for the detection of DNA and RNA in agarose gels. It is a highly sensitive and stable fluorescent dye that binds to nucleic acids, allowing visualization under ultraviolet or blue light illumination.
Automatically generated - may contain errors
Lab products found in correlation
Agarose, by Sangon (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Pcr purification kit, by Sangon (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Nicl2, by Sinopharm (1 mentions)
Exonuclease 3 exo 3, by New England Biolabs (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Cutsmart reaction buffer, by New England Biolabs (1 mentions)
Acetaldehyde, by Maclin Biochemical Technology (1 mentions)
L cystine, by Maclin Biochemical Technology (1 mentions)
L selenocystine, by Maclin Biochemical Technology (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Sirna, by GenePharma (1 mentions)
Hplc purified oligonucleotides, by Sangon (1 mentions)
Potassium chloride (kcl), by Sinopharm (1 mentions)
8 protocols using gelred
1
Electrophoretic Mobility Shift Assay (EMSA)
2
COVID-19 RNA Transcription and Detection
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To transcribe Se-RNA and S-RNA, DNA template (210 bp in N gene of COVID-19) was cloned, with T7 promoter, from pCOVID-19 by PCR (primer F: 5’-taatacgactcactatagCTCTTCTCGTTCCTCATC-3’; primer B: 5’-GCAGCAGATTTCTTAGTG-3’). Se-RNA and S-RNA were then transcribed with HiScribe T7 High Yield RNA Synthesis Kit by using all four NTPαSes and NTPαSs (SeNtInAll, Chengdu, China), respectively. After transcription, each product (1 μL) was analyzed by denaturing polyacrylamide gel electrophoresis (12% PAGE) and stained with GelRed (Sangon Biotech, Shanghai, China) and visualized by the ChemiDoc™ XRS Imaging System (Bio-Rad Co., California, USA).
3
DNA Sequence Synthesis and Purification
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The designed DNA sequences were synthesized and purified by GenScript Biotech Co., Ltd. (Nanjing, China). DNA strands modified with biotin were purified by High Performance Liquid Chromatography (HPLC), and other strands were purified by economic PAGE (ePAGE) or PAGE. The DNA strands are shown in Figure S1 . All sequences are shown in Table S1 . The concentration of each strand was estimated by measuring the UV absorbance at the wavelength of 260 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). STV (used without further purification), agarose, and GelRed were purchased from Sangon Bio-tech Co., Ltd. (Shanghai, China). NiCl2 was purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. All aqueous solutions were prepared with ultrapure water.
4
Gel Electrophoresis of DNA Solutions
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The DNA solutions were analyzed in an 8% native polyacrylamide
gel
in 1× TBE buffer after running for 70 min at a constant power
of 60 W using electrophoresis apparatus: DYCZ-20B (LIUYI BIOTECHNOLOGY
CO., LTD., China). Gels were stained with GelRed, which was purchased
from Sangon Biotech Co., Ltd. (Shanghai, China), and scanned with
a scanner (ProteinSimple, USA).
gel
in 1× TBE buffer after running for 70 min at a constant power
of 60 W using electrophoresis apparatus: DYCZ-20B (LIUYI BIOTECHNOLOGY
CO., LTD., China). Gels were stained with GelRed, which was purchased
from Sangon Biotech Co., Ltd. (Shanghai, China), and scanned with
a scanner (ProteinSimple, USA).
5
DNA Oligonucleotide Synthesis and Characterization
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All oligonucleotides used in this study were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). ALP, TdT, bovine serum albumin (BSA), exonuclease III (Exo III), T7 exonuclease (T7 exo), deoxyribonuclease I (DNase I), uracil glycosylase (UDG), CutSmart reaction buffer (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(Ac)2, 100 μg/mL BSA, pH 7.9, 25 °C), 10 × TdT buffer (500 mM KAc, 200 mM Tris-Ac, 100 mM Mg(Ac)2, pH 7.9), and 2.5 mM CoCl2 solution were purchased from New England Biolabs Inc. (Ipswich, MA, USA). dATP, agarose, loading buffer, DNA ladder mix, and nucleic acid dye GelRed were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The concentration of DNA oligonucleotides was measured using a NanoDrop 2000 UV–vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The sequences of all oligonucleotides are listed in Table S1 .
6
Optimizing siRNA Delivery for Apoptosis Studies
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L-cystine, L-selenocystine, acetaldehyde, and other chemicals/solvents were purchased from Macklin Lnc. (Shanghai, China). siRNA was synthesized by GenePharma. Lnc. The sequences used were from the literature: siRNA scramble control (siScr), 5′-CGUACGCGGAAUACUUCGATT[57 (link)]; BCL-xl siRNA sequence 5′-AUACUUUUGUGGAACUCUATT [58 (link)], BCL-w siRNA sequence 5′-GCAGACUUUGUAGGUUAUATT [59 (link)]. bPEI (branched Polyetherimide, molecular weight ~0.8 kDa), EDC (1-ethyl-3-(3-dimethy-laminopropyl) carbodiimide), and NHS (N-hydroxysulfosuccinimide) were purchased from Macklin Lnc. Fetal bovine serum (FBS). Trypsin-EDTA, penicillin-streptomycin (PS), DMEM cell culture medium, and phosphate buffered saline (PBS) were purchased from Gibco. TAE (tris-acetate), agarose, and Gel red were purchased from Sangon Biotechnology lnc. (Shanghai, China).
7
Sensitive Detection of Viral Nucleic Acids
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HPLC-purified oligonucleotides (Table S1† ) were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). ThT was purchased from Frontier Scientific, Inc. (Logan, USA). Potassium chloride was purchased from Sinopharm Group Chemical Reagent Co., Ltd (Shanghai, China). The gene segments from Hepatitis B and Ebola viruses and Nova virus (Table S2† ) were purchased from Jilin Kumei Biotechnology Co. Ltd. Taq PCR Master Mix for A-PCR, reaction buffers and GelRed were obtained from Sangon Biotech Co., Ltd (Shanghai, China). dNTP mix and enzymes (T4 DNA ligase and phi29 DNA polymerase) for RCA were obtained from New England Biolabs (Beijing, China). All the oligonucleotides were dissolved in Tris–HCl buffer (25 mM, pH 8.0), quantified using UV-vis absorption spectroscopy, stored at −20 °C, and annealed by heating at 95 °C for 3 min and gradually cooling to room temperature before use. ThT stock solutions (5 mM) were prepared in DMSO and stored in the dark at −20 °C. The human serum samples were provided by The First Bethune Hospital of Jilin University. Ultrapure water was utilized throughout.
8
OhrR Binding to Target Promoters
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The binding of OhrR protein or its mutated derivatives to target promoters was performed, as described previously [59 (link),60 (link)]. The promoter region of ohr was amplified and purified as DNA probe (181 bp). EMSA was performed by adding increasing amounts of purified OhrR protein to the DNA fragment (final concentration: 20 nM) in a total volume of 20 μL. The binding buffer contained 50 mM Tris-HCl (pH 8.3), 0.25 M KCl, 2.5 mM DTT, 5 mM MgCl2, 0.05 µg·mL−1 poly(dI-dC), 2.5 mM EDTA, 1% glycerol. The reaction mixtures were incubated for 20 min. at room temperature and then loaded onto a 6% native polyacrylamide gel in 0.5× Tris-borate-EDTA buffer at 150 V for 70 min. The gel was subsequently stained with GelRed (Sangon Biotech, Shanghai, China) for 20 min. and then imaged while using the gel imaging system.
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