As per the design of the RT stop assay, the RNA template is translated by the reverse transcriptase enzyme, up until it encounters a stable RNA G4 structure. Truncated complement DNA products are created and can be visualized by denaturing PAGE assay [44 (link)]. Texas Red-tagged primers were purchased from Sigma Aldrich, USA in lyophilized form and nuclease-free water was used to prepare 100 µM solutions. Each RT-stop experiment was performed in 10 μL reaction mixtures containing 2 μM RNA, 100 nM Texas Red-tagged primer, 2 mM NTPs and KCl/LiCl (150 mM). The tagged primer and RNAs were annealed by first denaturing by heating at 95 °C for 5 min, then cooling to room temperature over 2 h. Reverse transcriptase was added to the reaction and incubated for 1 h at 37 °C. The reverse-transcriptase reaction was stopped using a buffer consisting of 95% Formamide, 0.05% Bromophenol Blue, 20 mM EDTA, and 0.05% Xylene cyanol. The products were separated on a 15% denaturing (UREA) polyacrylamide gel, visualized on a ChemiDocTM MP Imaging system using the Rhodamine filter and then counter-stained with DiamondTM Nucleic Acid dye (Promega Corporation, San Luis Obispo, CA, USA) to visualize template bands.
Diamond nucleic acid dye
Manufactured by Promega
Sourced in United States
The Diamond™ Nucleic Acid Dye is a fluorescent stain used for the detection and quantification of nucleic acids in various applications, such as gel electrophoresis and real-time PCR. It binds to double-stranded DNA, single-stranded DNA, and RNA, emitting a fluorescent signal upon excitation that can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Typhoon trio scanner, by GE Healthcare (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Generuler 1 kb dna ladder, by Thermo Fisher Scientific (1 mentions)
Topvision agarose, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Promega (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Benchtop 1kb dna ladder, by Promega (1 mentions)
Blue orange loading dye, by Promega (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Qiamp dna mini kit, by Qiagen (1 mentions)
Mastercycler pro s, by Eppendorf (1 mentions)
Genejet whole blood genomic dna purification mini kit, by Thermo Fisher Scientific (1 mentions)
Infinite m1000 pro, by Promega (1 mentions)
Magnesium chloride, by Qiagen (1 mentions)
Coralload pcr buffer, by Qiagen (1 mentions)
Hotstartaq plus dna polymerase, by Qiagen (1 mentions)
Veriti, by Thermo Fisher Scientific (1 mentions)
100 bp dna ladder, by Thermo Fisher Scientific (1 mentions)
Alphaimager gel documentation system, by Bio-Techne (1 mentions)
29 protocols using diamond nucleic acid dye
1
Visualization of RNA G4 Structures via RT-Stop Assay
2
Magnetic Nanoparticles for Molecular Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The water used in the preparation of all solutions was ultra-pure. When not disclosed, reagents are from Fischer Scientific. A TRIS-EDTA (TE) buffer was prepared by combining TRIS, ethylenediaminetetraacetic acid (EDTA), and K2HPO4 at 10 mM, 1 mM, and 100 mM concentration, respectively. pH was adjusted to 7.4 using 1 M HCl. A TRIS-borate-EDTA (TBE) buffer 10x was acquired from FRILABO (Porto, Portugal). Phosphate buffer (PB) 0.1 M, pH 7.2 was prepared from stock solutions of Na2HPO4 and NaH2PO4 at 0.2 M. PB-Tween20 consisted on PB buffer with 0.02% (v/v) of Tween® 20 from Promega (Madison, WI, USA).
The customized single stranded oligonucleotide sequences were synthesized by STABVIDA (Caparica, Portugal).
DNA-free water, MasterMix 16S Basic, and Moltaq 16S from Molzym (Germany) were used for PCR reactions. Electrophoresis reagents included TopVision agarose from Thermo Fisher ScientificTM (MA, USA), for gel preparation, GRS DNA loading buffer blue 6x from GRiSP (Porto, Portugal), GeneRuler 1 kb DNA ladder from Thermo Fisher ScientificTM, and DiamondTM nucleic acid dye from Promega.
The MNPs were nanomag®-D from Micromod (Rostock, Germany), with a diameter of 250 nm and 75–80% (w/w) magnetite in a matrix of dextran (40 kDa), streptavidin coated. The particles have a magnetic moment of ∼1.6 × 10−16 Am2 for a 1.2 kA/m magnetizing field and a susceptibility of χ ∼ 4.
The customized single stranded oligonucleotide sequences were synthesized by STABVIDA (Caparica, Portugal).
DNA-free water, MasterMix 16S Basic, and Moltaq 16S from Molzym (Germany) were used for PCR reactions. Electrophoresis reagents included TopVision agarose from Thermo Fisher ScientificTM (MA, USA), for gel preparation, GRS DNA loading buffer blue 6x from GRiSP (Porto, Portugal), GeneRuler 1 kb DNA ladder from Thermo Fisher ScientificTM, and DiamondTM nucleic acid dye from Promega.
The MNPs were nanomag®-D from Micromod (Rostock, Germany), with a diameter of 250 nm and 75–80% (w/w) magnetite in a matrix of dextran (40 kDa), streptavidin coated. The particles have a magnetic moment of ∼1.6 × 10−16 Am2 for a 1.2 kA/m magnetizing field and a susceptibility of χ ∼ 4.
3
Phylogenetic Analysis of EF-1α Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For phylogenetic analysis, EF-1 α gene was amplified and sequenced using EF-1 and EF-2 primers (Table 2 ). Agarose gels of 0.8% concentration were made in 0.5× TBE (Tris borate EDTA) Buffer and run at 90 V, 100 mA for 60 min. Amplification products were compared to a 100 bp molecular weight marker, EZ Load™ 100bp Molecular Ruler (#170-8352) [52 ]). For gel staining, the 1× Diamond red intercalator, Diamond™ Nucleic Acid Dye from Promega, was used [53 ]. The gels obtained were visualized using a Gel Documentation System, UV light in Gel Doc EZ–Biorrad [54 ]. The amplified products were sequenced by the automatic pyro-sequencing method, Sanger dideoxy sequencing method, in Macrogen, Korea [55 ]. EF-1 α DNA sequences of 28 strains, together with known NRRL strains of FGSC from the GenBank, were aligned using ClustalW algorithm of MEGA 7.0.26 software [56 (link)]. The phylogenetic tree was constructed using the Maximum Likelihood method with 1000 bootstrap replicates. The best-fit model of molecular evolution was selected based on Bayesian Information Criterion scores [57 (link)].
4
Electrophoretic Mobility Shift Assay of MucR Protein Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EMSA experiments were carried out as previously reported [59 (link)].
Briefly, several amounts of the recombinant MucR purified from E. coli Bl21 (DE3) and of the naturally expressed MucR from S. meliloti 1021 were mixed with the DNA target sites tested in binding buffer (25 mM HEPES pH 7.9, 50 mM KCl, 6.25 mM MgCl2, 5% glycerol). Samples were incubated 10 min on ice and loaded in a 5% polyacrylamide gel. Electrophoresis was performed in 0.5× TBE and run at room temperature for 70 min at 200 V. Gels were stained for 20 min using Diamond™ Nucleic Acid Dye (Promega, Singapore) following the manufacturer’s instructions and imaged by Typhoon Trio+ scanner (GE Healthcare, Chicago, IL, USA).
For competition assays, competitors were added to the reaction mixture, prepared as described above, after proteins were incubated 10 min on ice with the FAM-labeled double-stranded oligonucleotides. After adding the competitors, samples were incubated for a further 10 min on ice and then loaded in a 5% polyacrylamide gel. Images were acquired by Typhoon Trio+ scanner (GE Healthcare) on the base of the FAM-labeled DNA fluorescence.
Protein amounts used, sequences, and amounts of double-stranded DNA targets are indicated in figures and figure legends.
Briefly, several amounts of the recombinant MucR purified from E. coli Bl21 (DE3) and of the naturally expressed MucR from S. meliloti 1021 were mixed with the DNA target sites tested in binding buffer (25 mM HEPES pH 7.9, 50 mM KCl, 6.25 mM MgCl2, 5% glycerol). Samples were incubated 10 min on ice and loaded in a 5% polyacrylamide gel. Electrophoresis was performed in 0.5× TBE and run at room temperature for 70 min at 200 V. Gels were stained for 20 min using Diamond™ Nucleic Acid Dye (Promega, Singapore) following the manufacturer’s instructions and imaged by Typhoon Trio+ scanner (GE Healthcare, Chicago, IL, USA).
For competition assays, competitors were added to the reaction mixture, prepared as described above, after proteins were incubated 10 min on ice with the FAM-labeled double-stranded oligonucleotides. After adding the competitors, samples were incubated for a further 10 min on ice and then loaded in a 5% polyacrylamide gel. Images were acquired by Typhoon Trio+ scanner (GE Healthcare) on the base of the FAM-labeled DNA fluorescence.
Protein amounts used, sequences, and amounts of double-stranded DNA targets are indicated in figures and figure legends.
5
Electrophoretic Mobility Shift Assay for Protein-DNA Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The electrophoretic mobility shift assay (EMSA) experiments were carried out as previously described [28 (link)]. In detail, protein amounts from 0.2 up to 1.6 μg were incubated for 10 min on ice with 5 pmol of the double-stranded oligonucleotides in binding buffer (25 mM HEPES pH 7.9, 50 mM KCl, 6.25 mM MgCl2, 5% glycerol). The reaction mixture of 20 μL was loaded onto a 5% polyacrylamide gel in 0.5X TBE and run at room temperature for 75 min at 200 V. Gels were then stained for 20 min with Diamond™ Nucleic Acid Dye (Promega), rinsed with milliQ water and imaged by Typhoon Trio+ scanner (GE Healthcare). In particular, the amount of MucR used in the EMSAs with babR60 was 0.2, 0.4 and 0.8 μg. In the EMSAs with all the other double-stranded oligonucleotides tested, the amount of MucR used was 0.4, 0.8 and 1.6 μg.
For competition assays, 5 pmol of the FAM-labeled double-stranded babR60 was used as a probe incubated for 10 min on ice with 0.4 μg of MucR. Then, 10X, 20X or 40X of each competitor was added and the reaction mixtures were incubated on ice for another 10 min. The electrophoreses were carried out as already describe above and the fluorescent signals were imaged by Typhoon Trio+ scanner (GE Healthcare).
For competition assays, 5 pmol of the FAM-labeled double-stranded babR60 was used as a probe incubated for 10 min on ice with 0.4 μg of MucR. Then, 10X, 20X or 40X of each competitor was added and the reaction mixtures were incubated on ice for another 10 min. The electrophoreses were carried out as already describe above and the fluorescent signals were imaged by Typhoon Trio+ scanner (GE Healthcare).
6
Electrophoretic Mobility Shift Assay of MucR Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The EMSA experiments were performed as previously described74 (link),75 (link). In detail, 0.4, 0.6 or 0.8 μg of each protein were incubated 10 min on ice in binding buffer (25 mM HEPES pH 7.9, 50 mM KCl, 6.25 mM MgCl2, 5% glycerol) with 5 pmol of double-stranded oligonucleotides Site 1(5′-GTTGCCTATTATTAATGTAATATGGTTTGA-3′) previously published as a target site of MucR and located at −174 bp from the ATG start codon of mucR gene27 (link). The protein/DNA ratio for each sample was 5, 8, 10 when 0.4, 0.6, 0.8 μg of protein were used respectively. To obtain a negative control, 0.8 μg of each protein were incubated with 5 pmol of the double-stranded oligonucleotide NC (5′-CGCGGCACGACCGCAGCGGTCGGGTGGCAC-3′) in the same binding buffer whose composition has been already described above. The total volume of each reaction mixture was 20 μl. After incubation on ice, the samples were loaded onto a 5% polyacrylamide gel in 0.5X TBE and run at room temperature for 70 min at 200 V. Gels were stained 20 minutes using Diamond™ Nucleic Acid Dye (Promega) following the manufacturer’s instructions and imaged by Typhoon Trio+ scanner (GE Healthcare). The results by EMSAs shown in this study are representative of more than 10 replicates.
7
Characterization of Pol Epsilon Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified wild-type Pol epsilon and dropout variants were pre-mixed in a dilution series (75, 150, 200, 300, and 450 nM) with DNA at a concentration of 300 nM, in a reaction buffer containing 25 mM HEPES, pH 8.0, 30% glycerol, 2 mM EDTA, 0.2 mg/ml bovine serum albumin (BSA), and 0.02% Triton-X. Binding was performed for 30 min at 4 °C. polyacrylamide gel electrophoresis (PAGE) made with 4% polyacrylamide, 3% glycerol, and 0.5× TAE polymerase was pre-run at 100 V for 1 h (4 °C). Protein–DNA complexes were resolved by running the PAGE at 100 V for ∼2 h (4 °C) in 0.5× TAE. Visualization of the complexes were done my staining the PAGE with Diamond Nucleic acid dye (Promega) for 15 min. Imaging was done with 1.5 s manual exposure using Bio-Rad Gel DOC XR+ equipped with the Bio-Rad image lab software.
8
Genetic Analysis of Asian Honeybee Species
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apis andreniformis was collected from Padang Pariaman, West Sumatra and A. c. indica was collected from Bogor, West Java, Indonesia. Total DNA was extracted from the thoraxes using a standard phenol–chloroform extraction method and ethanol precipitation (Sambrook et al. 1989 ), with minor modifications (Raffiudin & Crozier 2007 (link)).
The partial regions of PFK and PK-like gene primers were designed manually from A. mellifera (GenBank NC_007079, NC_007073), A. dorsata (GenBank NW_006263741, NW_006263478), and A. florea (GenBank NW_003790158, NW_003790664) genomic sequences. Due to an obstacle in primer design involving the 1,099 bp of Intron 3 in the A. mellifera PFK gene, the targeted gene was divided into two regions, Part A (exons 1–3) and Part B (exon 4–7) (Table 1 ). The PCR conditions were as follows: initial denaturing at 95°C for 3 min, 30 cycles of 95°C for 1 min, 48°C–53°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 2 min. PCR products were electrophoreses in 1.5% agarose gel and stained using Diamond Nucleic Acid Dye (Promega, Madison, WI, USA). The PCR products were sequenced by a company sequencing service (First BASE, Selangor, Malaysia).
The partial regions of PFK and PK-like gene primers were designed manually from A. mellifera (GenBank NC_007079, NC_007073), A. dorsata (GenBank NW_006263741, NW_006263478), and A. florea (GenBank NW_003790158, NW_003790664) genomic sequences. Due to an obstacle in primer design involving the 1,099 bp of Intron 3 in the A. mellifera PFK gene, the targeted gene was divided into two regions, Part A (exons 1–3) and Part B (exon 4–7) (
9
Human Topoisomerase I Relaxation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The inhibition of topoisomerase I relaxation activity was tested in triplicate with Human Topoisomerase I Relaxation High Throughput Plate Assay (Inspiralis Ltd., Norwich, UK) according to Vendor’s Protocol. Briefly, the test was based upon the measurement of fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The inhibition of human Topo I relaxation activity was measured in assay mixture containing Topo I and supercoiled DNA substrate (plasmid pNO1). At the first step—the activity of Topo I—the amount of enzyme needed to fully relax the plasmid was determined. Then, for this specific amount of enzyme. the relaxation inhibition potency of the studied compound was tested. The increasing amounts of compounds (2–100 µM) were added to the reaction mixture together with the positive control and reference standard: camptothecin and SN38, respectively. The fluorescence of DNA plasmid stained with Diamond™ Nucleic Acid Dye (Promega, Madison, WI, USA) was read in fluorescence plate reader (495 nm/537 nm) [36 (link),37 (link)].
10
Dendrimer-DNA Complexation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dendrimers/DNA complexes
at different N/P ratios (5, 15, and 30) were prepared following complexation
instructions of the transfection protocol. 250 ng of pGFP or model
DNA (herring sperm for proof-of-concept assays), both from Promega,
was used to form complexes with a proper dilution of the dendrimer
in deionized water. Complexes were then diluted to 20 μL final
volume, and 4 μL of loading dye (blue/orange loading dye, 6×
from Promega) was added to dendriplexes. 24 μL of each sample
was loaded on 0.8% (w/v) agarose gel. DNA ladder (BenchTop 1 kb DNA
Ladder, from Promega) was loaded in the first lane, while free DNA
was loaded in the last one. Electrophoresis running was performed
in Tris-acetate-EDTA buffer (1× TAE) at 150 V for 30 min. The
gel was subsequently placed into a plastic tray and incubated with
staining solution (Diamond Nucleic Acid Dye Promega diluted in 1×
TAE buffer) following the manufacturer’s instructions, for
20 min under gentle shaking, protected from light. The gel was analyzed
under UV Transilluminator BIO-RAD ChemiDoc XRS+ using a proper filter.
at different N/P ratios (5, 15, and 30) were prepared following complexation
instructions of the transfection protocol. 250 ng of pGFP or model
DNA (herring sperm for proof-of-concept assays), both from Promega,
was used to form complexes with a proper dilution of the dendrimer
in deionized water. Complexes were then diluted to 20 μL final
volume, and 4 μL of loading dye (blue/orange loading dye, 6×
from Promega) was added to dendriplexes. 24 μL of each sample
was loaded on 0.8% (w/v) agarose gel. DNA ladder (BenchTop 1 kb DNA
Ladder, from Promega) was loaded in the first lane, while free DNA
was loaded in the last one. Electrophoresis running was performed
in Tris-acetate-EDTA buffer (1× TAE) at 150 V for 30 min. The
gel was subsequently placed into a plastic tray and incubated with
staining solution (Diamond Nucleic Acid Dye Promega diluted in 1×
TAE buffer) following the manufacturer’s instructions, for
20 min under gentle shaking, protected from light. The gel was analyzed
under UV Transilluminator BIO-RAD ChemiDoc XRS+ using a proper filter.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!