The inhibition of yeast topoisomerase II was analysed according to the relaxation assay kit from Inspiralis (Inspiralis Limited, Norwich, UK). Briefly, 250 ng of supercoiled pBR322 DNA (Thermo Fisher Scientific, Waltham, MA, USA), 1 mM ATP (Inspiralis Limited, UK), and 1–200 μM of the analysed compound were mixed with reaction buffer (1 mM Tris-HCl (pH 7.9), 10 mM KCl, 0.5 mM MgCl2, 0.2 % (by volume) glycerol). The reaction was initiated by the addition of an enzyme, allowed to proceed at 30 °C for 30 min and terminated by addition of 40% (m/V) sucrose, 100 mM Tris-HCl pH 8, 10 mM EDTA, and 0.5 mg mL−1 Bromophenol Blue. Two-step extraction with chloroform:isoamyl alcohol (24:1, by volume) and butanol water was performed and mixtures were analysed on 1% agarose gel in 1 × TAE buffer, 3h, 4.5 V cm−1. Gel was stained in GelRed 3 × staining solution (Biotium, Fremont, CA, USA) for 30 min and photographed with Gel Doc XR+Gel Documentation System (Bio-Rad: Hercules, CA, USA).
Pbr322 dna
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PBR322 DNA is a small, high-copy-number plasmid commonly used as a cloning vector in molecular biology. It contains an origin of replication and a selectable antibiotic resistance marker, allowing for the propagation and selection of recombinant DNA constructs in bacterial hosts.
Automatically generated - may contain errors
Lab products found in correlation
Gelred 3 staining solution, by Biotium (1 mentions)
Gel doc xr gel documentation system, by Bio-Rad (1 mentions)
Sulfanilic acid, by Merck Group (1 mentions)
Sodium nitrite, by Merck Group (1 mentions)
Ferrous sulfate heptahydrate feso4, by Merck Group (1 mentions)
Folin ciocalteau reagent, by Merck Group (1 mentions)
1 1 diphenyl 2 picrylhydrazyl dpph, by Fujifilm (1 mentions)
Dna loading dye, by Thermo Fisher Scientific (1 mentions)
Raw 264.7 cell, by Korean Cell Line Bank (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Gallic acid, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
Puc19 dna, by Thermo Fisher Scientific (1 mentions)
Crrna, by Integrated DNA Technologies (1 mentions)
Milli q system, by Merck Group (1 mentions)
Safe imager 2, by Thermo Fisher Scientific (1 mentions)
4 protocols using pbr322 dna
1
Topoisomerase II Inhibition Assay
2
Antioxidant Assays and DNA Protection
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Folin-ciocalteau (FC reagent), gallic acid, 2,2-azino-bis (3-ethylbenzo thiazoline-6-sulfonic acid) diammonium salt (ABTS), sodium nitrite, sulfanilic acid, acetic acid, sodium citrate, ascorbic acid and ferrous sulfate heptahydrate (FeSO4) were obtained from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). D-Catechin was purchased from MP Biomedicals, LLC (Illkich, France). Hydrogen peroxide 30% was obtained from Daejung Chemicals and Metals Co. (Gyeonggi-do, Korea). pBR322 DNA and 6× DNA loading dye were purchased from Thermo Fisher Scientific Inc. (NYSE: TMO, Waltham, MA USA; Gyeonggi-do, Korea). RAW 264.7 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). DMEM, penicillin-streptomycin and fetal bovine serum (FBS) were purchased from GIBCO BRL (Life Technologies, Grand Island, NY, USA). All chemicals were used without any further purification.
3
LbaCas12a Nuclease Cleavage Assay
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LbaCas12a, crRNA, synthetic target, and crRNA were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA). rCutSmart™ buffer, and NEBuffer™ 2.1, pUC19 DNA (Catalog# FERSD0061), pBR322 DNA (Catalog# FERSD0041 and phiX174 DNA (Catalog# FERSD0031), were purchased from Thermo-fisher. Ultrapure water (18.3 MΩ cm) was produced by the Milli-Q system (Millipore, Inc., USA) and used throughout the experiments. The sequences of oligonucleotides and crRNA molecules are listed in Supplementary Table 1.
4
DNA-Ligand Binding Assay Using Agarose Gel Electrophoresis
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Assays were performed using 1.2% (w/v) agarose gels. A plasmid pBR322 DNA was purchased from Thermofisher Scientific® at 0.5 μg μL−1 concentration in phosphate buffer 50 mM (pH 7.4). A 20 μL portion containing 0.125 μg μL−1 of DNA in 10 mM Tris-HCl (pH 7.6) and 1 mM EDTA was incubated with increasing volumes of the stock solution of 11Cl (1 mM in DMSO) at ri values ranging from 0.05 to 0.5 (ri = [complex]/[no. nucleotide]). Samples were incubated at 37 °C for 24 h, after which 2 μL of the TriTrack DNA loading dye buffer was added. 20 μL of the sample was loaded in the agarose gel (1.2% w/v), and electrophoresis was carried out for a period of 100 min at 40 V in TAE 1× (Tris-acetate/EDTA) buffer. After electrophoresis, the gel was immersed in 200 mL of TAE 1× buffer containing 20 μL of a stock solution of SYBR safe for 30 min to stain the DNA. Then, the gel was washed with Mili Q water for 10 min and the stained gel was analyzed with a blue light in a Safe Imager™ 2.0 (Invitrogen).
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