The fluorescence analysis was performed in a black microplate (384-well, Corning, USA) on a Multifunctional Microplate Reader (Varioskan Flash, Thermo Scientific, USA). The excitation wavelength was 350 nm. All of the reactions were carried out in 4 mM BPES buffer (pH = 7) at room temperature, as described previously31 (link). Blank buffer, RNA, DNA and BBR alone were also measured as background and controls. Double-stranded DNA oligos were obtained from mix of complementary oligos (equal molar concentrations) by denaturing at 95 °C for 5 min and then slow cooling to room temperature over a period of 1 h. The DNA oligonucleotides were synthesized by Sangon Biotech (Shanghai, China) and the RNA oligos with poly (A) tails by Sigma Aldrich (USA). The sequences of all DNA and RNA oligos used in these experiments are shown in Supplementary Tables 1 & 2 .
Dna oligonucleotides
Manufactured by Merck Group
Sourced in United States, Japan
DNA oligonucleotides are synthetic, short, single-stranded DNA molecules that are commonly used as building blocks in various molecular biology and genetic engineering applications. They are designed to have a specific sequence of nucleotides and can be utilized for a range of purposes, such as gene expression analysis, DNA sequencing, and PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
T4 ligase, by Thermo Fisher Scientific (11 mentions)
Turbofect, by Thermo Fisher Scientific (10 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (9 mentions)
γ 32p atp, by PerkinElmer (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Genelute hp plasmid miniprep kit, by Merck Group (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Restriction enzyme, by Thermo Fisher Scientific (6 mentions)
T4 polynucleotide kinase, by New England Biolabs (6 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (5 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (5 mentions)
Hifi assembly master mix, by New England Biolabs (5 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (5 mentions)
Agencourt ampure xp bead, by Beckman Coulter (5 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (5 mentions)
Bioruptor 0.5 ml microtubes for dna shearing, by Diagenode (5 mentions)
Transcriptaid t7 high yield transcription kit, by Thermo Fisher Scientific (4 mentions)
Rna clean concentrator kit, by Zymo Research (4 mentions)
Zymopure plasmid midiprep kit, by Zymo Research (4 mentions)
Neb5 alpha competent cells, by New England Biolabs (4 mentions)
53 protocols using dna oligonucleotides
1
Fluorescence analysis of DNA and RNA interactions
2
Cloning and Purification of Gbp2 and Hrb1
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Gbp2 and Hrb1 coding sequences were cloned from Saccharomyces cerevisiae genomic DNA by PCR using Hifi KOD DNA polymerase (Novagen) and a library of DNA oligonucleotides (Sigma, IDT). Amplified products were purified, digested with corresponding restriction enzymes and ligated into a home-modified pET28 (Novagen) vector that contains: a thioredoxin A N-terminal tag to enhance expression, a 6xHis tag for purification, and a TEV site for fusion removal. Bacterial plasmids used in this work are summarized in Supplementary Figure S1.
3
Reagents for Molecular Biology Experiments
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Inorganic salts, acids, bases, and organic compounds were purchased from Merck (Kenilworth, NJ, USA), Sigma Aldrich (St Louis, MO, USA), and AppliChem (Darmstadt, Germany). Chromatography resins for protein purification used in this work were Ni-NTA Superflow purchased from Qiagen (CITY, Germany) and Heparin Sepharose Fast Flow to GE Healthcare (Fairfield, CT, USA). Ultrapure NTPs, dNTPs, and ddNTPs were supplied by GE healthcare (Fairfield, CT, USA). Labelled nucleotides and [α-32P]dGTP were purchased from Perkin Elmer (Waltham, MA, USA). T4 polynucleotide kinase for DNA labelling was obtained from New England Biolabs (Ipswich, MA, USA) and used as indicated by the manufacturer. Vent polymerase was supplied by New England Biolabs (Ipswich, MA, USA). DNA oligonucleotides were synthesized by Sigma Aldrich (St Louis, MO, USA) and purified by 8 M urea-20% polyacrylamide gel electrophoresis. Oligonucleotides containing 8-oxoG were purchased from Eurogentec (Seraing, Belgium).
4
Electrochemical DNA Biosensor Fabrication
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A glassy carbon electrode (3 mm diameter) was obtained from BASi® (Lafayette, USA). All DNA oligonucleotides were synthesised by Sigma-Aldrich Co. Ltd. (Gillingham, UK). Potassium ferri/ferrocyanide, bovine serum albumin (BSA) and Tris HCl were obtained from Sigma-Aldrich. RT-PCR materials were purchased from Qiagen® (Manchester, UK). HPLC purified RNA was purchased from IDT® (Leuven, Belgium), the remaining buffer ingredients and 1-amino-2-naphthol-4-sulfonic acid were purchased from Fisher Scientific® (Loughborough, UK). RNase free water was produced by overnight treatment with 0.1% v/v DEPC and subsequent autoclaving. The DNA and RNA were dissolved in RNase free pH 8.0 TMD buffer consisting of 50 mM Tris-HCl, 20 mM MgCl2 and 1 mM dithiothreitol. DNA and RNA samples were stored frozen at −80 °C and denatured at 80 °C for 2 min prior to use, sequence data are provided in the supplementary information (Supplementary table S1).
5
Primer Design for RT-qPCR Analysis
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DNA oligonucleotides (Sigma-Aldrich; Table S6 for RT-qPCR were designed using Clone Manager 9 Professional Edition or Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi ).
6
Plasmid Construction and Purification
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Restriction enzymes, T4 ligase, TranscriptAid T7 High Yield Transcription Kit, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, Turbofect and Penicillin/Streptomycin were purchased from Thermo Fischer Scientific. DNA oligonucleotides and the GenElute HP Plasmid Miniprep kit used in plasmid purifications were acquired from Sigma-Aldrich. T4 Polynucleotide Kinase, Q5 High-Fidelity DNA Polymerase, NEB Stable Competent E. coli, HiFi Assembly Master Mix were from New England Biolabs Inc. NucleoSpin Gel and PCR Clean-up kit used to clean up DNA from agarose gels were purchased from Macherey-Nagel. ZymoPURE Plasmid Midiprep Kit and RNA Clean & Concentrator Kit were from Zymo Research. Ampicillin, kanamycin and chloramphenicol antibiotics were from Sigma-Aldrich, NaCl, yeast extract and tetracycline were from Molar chemicals, agarose and tryptone were from VWR.
7
Binding Assay for G-quadruplex Ligands
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Small-molecule ligands, namely Braco-19 and TMPyP4, were purchased from Sigma Aldrich Chemicals, dissolved in water or DMSO, respectively, for preparation of stock solutions, and stored at 4°C until further use. Other reagents and chemicals including KCl, KH2PO4, K2HPO4, DMSO, and D2O were procured from Sigma Aldrich Chemicals or HiMedia Laboratories. The DNA oligonucleotides and the RNA oligonucleotides used in the studies were obtained from Sigma Aldrich Chemicals and IDT Technologies, respectively (Table S8 ).
8
KRAS Gene Mutation Cloning and Verification
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DNA oligonucleotides were purchased from Sigma-Aldrich (Ishikari, Japan), and stored as 100 mM stock solutions in 10 mM Tris-HCl with 1 mM EDTA (pH 8.0). WT human genomic DNA was purchased from Promega (Tokyo, Japan). Heterozygote human genomic DNA with seven mutations (G12A, G12C, G12D, G12R, G12S, G12V and G13D) in the KRAS gene codon 12 and 13 were obtained from Horizon Diagnostics (Cambridge, UK). Eprobes (shown in Table I ) were obtained from K.K. DNAFORM (Yokohama, Japan). Point mutations in codon 12 of the KRAS gene (GAT, GCT, GTT, AGT, TGT) were cloned into the plasmid pGEM-T (Promega) as previously described (26 (link)) and stored in glycerol at −80°C for long-term conservation. Point mutation of codon 13 (GAC) in the KRAS gene was prepared using the same protocol. The KRAS codon 12 (CGT) point mutation was prepared using the synthetic DNA ordered and inserted into a pUC57 plasmid (GenScript, Tokyo, Japan). The sequences of WT and all seven mutant clones were verified on ABI PRISM 3100 Avant (Applied Biosystems, Tokyo, Japan), diluted in TE buffer and stored at −20°C until use.
9
Radiolabeling of DNA Oligonucleotides
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DNA oligonucleotides were synthesized by Sigma Aldrich (St Louis, Mo, USA) and Invitrogen (La Jolla, CA, USA). Oligonucleotides containing 8oxoG were purchased from Eurogentec (Seraig, Belgium). The synthetic AP nucleotide was obtained from LinkTech and used to customize oligonucleotide synthesis. Unlabeled ultrapure dNTPs were supplied by GE (Fairfield, CT, USA). T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) was used for 5′-oligonucleotide labeling with [γ-32P] ATP from Perkin Elmer (Waltham, MA, USA).
10
HPLC-Purified RNA Oligonucleotide Synthesis
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The HPLC-purified RNA oligonucleotides
(Table 1 ) were purchased
from Sigma-Aldrich. The concentration of oligonucleotides was calculated
from their absorbance readings obtained from the UV spectrophotometer
and their molar extinction coefficient values.
The DNA oligonucleotides employed
for the cloning purpose were
also procured from Sigma-Aldrich.
(
from Sigma-Aldrich. The concentration of oligonucleotides was calculated
from their absorbance readings obtained from the UV spectrophotometer
and their molar extinction coefficient values.
The DNA oligonucleotides employed
for the cloning purpose were
also procured from Sigma-Aldrich.
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