A part of PCR products (7.0 μL) were mixed with 1.0 μL DNA gel loading dye (6X) (Thermo Scientific) and the mixtures were then loaded to different lanes of an agarose minigel (5%) and run at 80 V for 90 min. The 5% agarose minigels were freshly prepared each time before application using 1X TBE buffer, and stained by SYBR Safe dye. TBE buffer (1X) was also used as running buffer. After separation of DNA fragments of the PCR products, the gel images were recorded using a Quantum VilberLourmat gel documentation system. A 50 bp DNA ladder (Invitrogen, Cat. number: 10416014) was used as DNA size marker to determine the band sizes.
Dna gel loading dye 6x
Manufactured by Thermo Fisher Scientific
Sourced in United States
DNA gel loading dye (6X) is a solution used to prepare DNA samples for electrophoresis. It increases the density of the sample, allowing it to sink into the gel, and contains a tracking dye to visually monitor the DNA migration during the electrophoresis process.
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Lab products found in correlation
Biodoc analyzer, by Analytik Jena (2 mentions)
Peqgreen, by Avantor (2 mentions)
Eos 1100d, by Canon (2 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
Hd green plus dna stain, by Intas (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Isogen Life Science (1 mentions)
Blood tissue kit, by Qiagen (1 mentions)
Quantifluor dsdna system, by Promega (1 mentions)
Mag bind total pure ngs kit, by Omega Bio-Tek (1 mentions)
Horizontal electrophoresis unit, by Bio-Rad (1 mentions)
Du series 600 spectrophotometer, by Beckman Coulter (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Generuler low range dna ladder, by Thermo Fisher Scientific (1 mentions)
Ls reloaded scanner, by Tecan (1 mentions)
Cholesterol, by HiMedia (1 mentions)
100 bp dna ladder, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by HiMedia (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ethanol, by Sisco Research Laboratories (1 mentions)
Isopropanol, by Sisco Research Laboratories (1 mentions)
9 protocols using dna gel loading dye 6x
1
Agarose Gel Electrophoresis of PCR Products
2
Agarose Gel Electrophoresis of DNA
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The extracted DNA was analyzed using agarose 0.8% gel electrophoresis separation. Fifteen microliters of DNA extract were mixed with 5 µl DNA gel loading dye (6X) (Thermo Scientific, Waltham, USA). The separated DNA bands were compared with a standard 1 Kb DNA extension ladder marker (Thermo Scientific, MA, USA).
3
Leptospira Species Identification by secY PCR
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For samples with a Ct value > 36 or with incomplete MLST results, a conventional PCR targeting the secY gene (657 bp) was performed to determine Leptospira species as previously described [54 (link)]. As positive control, DNA of a laboratory strain of L. interrogans serovar Icterohaemorrhagiae was used [55 (link)].
PCR products were prepared with DNA Gel Loading Dye (6x) (Thermo Fisher Scientific, Darmstadt, Germany) for gel electrophoresis in 2% agarose, and gels were stained with HDGreen Plus DNA Stain (Intas Science Imaging Instruments GmbH, Göttingen, Germany). Amplification products were visualised by UV light using the UVP GelSolo streamlined gel documentation (Analytik Jena AG, Jena, Germany). The samples were purified for sequencing using a NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) as recommended by the manufacturer. The sequences were trimmed using Bionumerics v.7.6.1. (Applied Maths Inc., Austin, TX, USA) and compared to available data in GenBank with the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi , accessed on 7 August 2022). The obtained sequences were uploaded to GenBank (accession numbers: OQ865429–OQ865435).
PCR products were prepared with DNA Gel Loading Dye (6x) (Thermo Fisher Scientific, Darmstadt, Germany) for gel electrophoresis in 2% agarose, and gels were stained with HDGreen Plus DNA Stain (Intas Science Imaging Instruments GmbH, Göttingen, Germany). Amplification products were visualised by UV light using the UVP GelSolo streamlined gel documentation (Analytik Jena AG, Jena, Germany). The samples were purified for sequencing using a NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) as recommended by the manufacturer. The sequences were trimmed using Bionumerics v.7.6.1. (Applied Maths Inc., Austin, TX, USA) and compared to available data in GenBank with the Basic Local Alignment Search Tool (BLAST) (
4
Genomic DNA Extraction from Cell Suspensions
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One milliliter of the cell suspensions was transferred into sterile 2 mL tubes. Cells were pelleted by centrifugation (10,000 RCF × 15 min), supernatant was discarded, and cells were lysed by homogenization into 180 μL of lysis buffer (1 × Tris-EDTA buffer pH = 8, 1.2% Triton X-100 and 20 mg mL−1 lysozyme in Rnase-free water) and incubated at 37 °C for 30 min. Subsequently, 25 μL of proteinase K and 200 μL of AW buffer from the Qiagen Blood & Tissue kit (Qiagen, Venlo, The Netherlands) were added, followed by an incubation at 56 °C for 30 min. The gDNA isolation protocol of the Qiagen kit was used. The DNA purity (260/280 nm and 260/230 nm) was assessed with a NanoDrop ND-1000 spectrophotometer (Isogen Life Science, The Netherlands), the DNA concentration was quantified using the QuantiFluor® dsDNA System (Promega, The Netherlands), and its integrity was assessed. DNA gel-loading dye (6X) by Thermo Fisher (US) was used before loading the samples in an agarose gel 1.5% (120 V; 45 min). Finally, gDNA samples were stored at −20 °C for further analyses.
5
Gel Electrophoresis for RPA Efficiency
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For the detection of the RPA efficiency via gel electrophoresis, the products were purified with the Mag-Bind® Total Pure NGS Kit (Omega Bio-Tek; M1378-01) according to the manufactures instructions with a finishing eluation step in 15 µl ddH2O and 3 µl DNA Gel Loading Dye (6X) (Thermo Fisher Scientific; R0611) was added. An agarose gel with a 2% concentration in 1 × TAE-buffer (50 × TAE-buffer; PanReac AppliChem; A1691) was used for the separation of the fragments containing 2 µl of peqGreen (VWR; 732–3196) in a volume of 50 ml gel to visualize the band. A total volume of 18 µl of the purified and prepared samples were used for the electrophoresis which ran at 120 V for 1 h before the detection via BioDoc Analyzer (Biometra) including a Canon EOS 1100D was made.
The detection of the Cy5-dUTP labelled amplicons via microarray was performed without any purification of the RPA products.
The detection of the Cy5-dUTP labelled amplicons via microarray was performed without any purification of the RPA products.
6
Radiation-Induced Apoptosis in Murine Cells
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The experiment was designed to study the radiation induced apoptosis in the form of DNA ladder in blood and bone maarow cells of mice in different treatment group at 6 h time point. The RBC lysed cells were fixed with 70% ethanol and stored overnight at -20°C. Total DNA was isolated from the ethanol fixed 1 × 106 cells using “DNeasy Blood & Tissue Kits” acquired from QIAGEN (according to the manufacturer’s protocol). DNA quantification was done using UV visible spectrophotometer (DU Series 600 spectrophotometer) from Beckman Coulter. Five microgram of extracted DNA was mixed with 3 μl of DNA Gel Loading Dye (6X) from Thermo Fisher Scientific and electrophoresed on a 1.2% agarose gel at 100 v for 30 min on Biorad Horizontal electrophoresis unit. The gel was visualized by UV light after standard ethidium bromide staining. The gels were analyzed by Gel-analyzer software.
7
Gel Electrophoresis of Purified DNA
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For gel electrophoretic analysis, 3 µl DNA Gel Loading Dye (6 x) (Thermo Fisher Scientific; R0611) were added to the purified (and digested) samples except the RsaI approach. For band visualization, a 2% agarose gel was prepared in 1 × TAE buffer (50 × TAE buffer; PanReac AppliChem; A1691) containing 4 µl peqGreen (VWR; 732-3196) per 100 ml gel. 12–15 µl of the purified and prepared samples were used for electrophoresis at 120 V for 1 h before detection via BioDoc Analyzer (Biometra) using a Canon EOS 1100D. GeneRuler Low Range DNA Ladder (Thermo Fisher Scientific; SM1191) and peqGOLD DNA ladder (VWR; 25-2040) were used for calculation of fragment length.
For the detection of incorporated Cy5-dUTP the DNA gel was scanned with a Tecan LS reloaded scanner using a 3D-printed gel tray.
For the detection of incorporated Cy5-dUTP the DNA gel was scanned with a Tecan LS reloaded scanner using a 3D-printed gel tray.
8
Comparative Cytotoxicity in A549 and V79 Cells
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A549 adenocarcinoma human alveolar basal epithelial cells and V79 Chinese hamster lung fibroblasts were purchased from the National Centre for Cell Science (NCCS), Pune. Dulbecco’s Modified Eagle’s Medium (DMEM) and antibiotic solution were purchased from HiMedia, Mumbai, India. Fetal bovine serum (FBS) was purchased from Gibco, Waltham, MA, USA. Other chemicals such as MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, acridine orange, ethidium bromide, and cholesterol were purchased from HiMedia, India. The 100 bp DNA ladder and DNA gel-loading dye (6x) were purchased from Thermo Fisher Scientific, Mumbai, India. Dimethyl sulfoxide (DMSO), chloroform, isopropanol, and ethanol were purchased from SRL chemicals, Mumbai, India.
9
Nanoparticle-based Metformin Delivery System
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