Purple loading dye

Manufactured by New England Biolabs

Sourced in United Kingdom

6× purple loading dye is a buffer solution used in gel electrophoresis to visually track the migration of DNA, RNA, or protein samples during the electrophoresis process. It contains a purple dye that allows the user to monitor the progress of the separation.

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Lab products found in correlation

Sybr gold, by Thermo Fisher Scientific (5 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) T7 endonuclease, by New England Biolabs (1 mentions) Neb buffer 2, by New England Biolabs (1 mentions) Ecori restriction enzyme, by New England Biolabs (1 mentions) Sybr safe dye, by Thermo Fisher Scientific (1 mentions) 9w lamp, by Philips (1 mentions) Chemdoc, by Bio-Rad (1 mentions) Typhoon gel scanner, by GE Healthcare (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) 1 kb plus dna ladder, by New England Biolabs (1 mentions) Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions) Amersham imager 600, by GE Healthcare (1 mentions) Nucleospin plasmid dna purification kit, by Macherey-Nagel (1 mentions) Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (1 mentions) Genelute bacterial genomic dna kit, by Merck Group (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions) Ibright 1500 imaging system, by Thermo Fisher Scientific (1 mentions) Pet28b, by New England Biolabs (1 mentions)

Close spelling variants of this product

Gel Loading Dye Purple (6×) Purple Gel Loading Dye Purple Loading Dye without SDS 6× loading dye

13 protocols using purple loading dye

Optimized DNA Extraction and Visualization

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DNA was eluted out of pellets for subequent analysis using gel electrophoresis by removing the supernatant after centrifugation, rinsing the pellet twice with cold 50% ethanol, and resuspension in TE (10 mM Tris [pH 8.0] + 1 mM EDTA). Elution efficiency was improved by increasing the concentration of metal-chelating EDTA (10 mM and 50 mM) and by heating the samples at 42°C or 65°C for 5 min. Samples were mixed with purple loading dye (New England Biolabs) prior to electrophoresis.
Gel electrophoresis experiments were performed using 1.0% agarose gels run in 1 x TAE using Horizon gel systems (LabRepco) as described [32 (link)]. Gels were stained with ethidium bromide and photographed using an Alpha Innotech RED gel imagestation.

England C.J., Gray T.C., Malla S.R., Oliveira S.A., Martin B.R., Beall G.W, & Lewis L.K. (2020). pH-dependent sedimentation of DNA in the presence of divalent, but not monovalent, metal ions. Analytical biochemistry, 616, 114099.

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T7 Endonuclease Assay for DNA Mutation Detection

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PCR products were purified (Qiagen, Cat: 28104) and eluted in molecular biology grade water and were then adjusted to 1X NEB Buffer 2.1 (NEB, Cat: B7202S). Products were then boiled for 10 minutes, allowed to cool to room temperature, divided in two, and treated with 1.5ul of T7 Endonuclease (NEB, Cat: M0302S) or water. The reaction proceeded for 1hr, until it was deactivated by adjusting the mixture with 1X Purple loading dye (NEB, Cat: B7024S). The reaction was then run at 120V for 65 minutes on a 1.5% agarose gel. The band intensities were quantified by densitometry using image-j according to previously published procedures [16 (link)]. For fragment analysis we utilized the method by Ran et al [16 (link)]. Results represent two to three independent experiments and error bars reflect standard deviation from the mean.

Huhn S.C., Ou Y., Kumar A., Liu R, & Du Z. (2019). High throughput, efficacious gene editing & genome surveillance in Chinese hamster ovary cells. PLoS ONE, 14(12), e0218653.

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Investigating Nucleosome-Transcription Factor Interactions

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3 mg wild-type CX3CR1 nucleosome array or mutated nucleosome array or 601 nucleosome array in 20 μL was mixed with PU.1 or ^DBDC/EBPα with a molar ratio to the nucleosome array ranging from 0:1 to 16:1, respectively, in digestion buffer (10 ul, 10 mM Tris-HCl, pH 8.0, 60 mM NaCl, 1 mM magnesium chloride, 2 mM DTT) at room temperature. 10 units of restriction enzyme XhoI (NEB) were added to the nucleosome array with and without PU.1. 5 units of restriction enzyme EcoRI (NEB) were added to the nucleosome array in the absence and presence of ^DBDC/EBPα. Samples were incubated at 37 °C for 30 min, and the enzyme was inactivated by NEB purple loading dye. Samples were then incubated with proteinase K at 50 °C for 60 min. After centrifugation, the top solution was harvested and loaded into a 1 % agarose gel stained with SYBR Safe dye (Invitrogen). Electrophoresis was performed at 130 V in 1 × TBE buffer for 25 min. Band intensities for the digestion product and input were measured using ImageJ. The relative digestion efficiency was calculated using the intensity of the product in the presence of transcription factors divided by the product intensity without transcription factors.

Lian T., Guan R., Zhou B.R, & Bai Y. (2023). Structural mechanism of synergistic targeting of the CX3CR1 nucleosome by PU.1 and C/EBPα. bioRxiv.

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Plasmid DNA Photocleavage Assay

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The cleavage of pUC19 DNA (0.2 μg) was studied in 1% agarose gel, where electrophoresis was done for 1 hr at 50 V using 1X TAE (Tris-acetate EDTA) running buffer (pH 8.3) [30 ]. For DNA photo-cleavage studies, the reactions were carried out under UVA light (Philips, 9W lamp, dose rate 6.198 J/m-1/s). The experiments were performed in a total volume of 20 μl that contained plasmid DNA (0.2 μg) in 50 mM Tris HCl buffer (pH 7.2) with 50 mM NaCl; according to the need of the experiment, ACPH (8 μM) was present. Then, the samples were irradiated with UVA light (15 KJ/m²) and analyzed for the photo-cleaved product formation in gel electrophoresis. 1X Purple loading dye (#B7024S; NEB) was mixed with each reaction mixture. The agarose gel was stained with ethidium bromide (EtBr) (0.2 μg/ml) after the run, visualized and photographed using ChemDoc™ in Bio-Rad XRS+.

Hansda S., Ghosh G, & Ghosh R. (2020). 9-phenyl acridine photosensitizes A375 cells to UVA radiation. Heliyon, 6(9), e04733.

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Alkaline Gel Electrophoresis of DNA

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Samples were prepared for alkaline gel electrophoresis in 6x Purple Loading Dye (NEB). Agarose gels (0.4%) were prepared in alkaline buffer (50 mM NaOH, 10 mM EDTA) and electrophoresis was performed in the same buffer at 1 V cm^-1 and 4 °C for 16 hours with buffer circulation. Gels were neutralized in 50 mM Tris pH 7.5 buffer, stained with SYBR Gold (Thermo Fisher) and visualized on a Typhoon gel scanner (GE Healthcare).

Nye D.B, & Tanner N.A. (2022). Chimeric DNA byproducts in strand displacement amplification using the T7 replisome. PLoS ONE, 17(9), e0273979.

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DNA End Processing by SOX Proteins

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Reactions (80 ul) consisted of 250 nM PstI-linearized 5.4 kB pGEX-6p DNA (500 nM DNA ends) or 250 nM of non-linearized plasmid, 25 nM WT SOX, 20 mM HEPES (pH 7.2), 3 mM MgCl₂, 0.5 mM TCEP, 70 mM NaCl. Each reaction was conducted in a 30°C water bath. 10 μl of each reaction was removed at the indicated times and quenched by adding 2 μl of 250 mM EDTA and 3 μl 6X purple loading dye (New England Bio Labs). Samples were loaded onto a 0.8% agarose gel stained with a 1/10 000 dilution of SYBR gold (Invitrogen) and visualized using a ChemiDoc MP imaging system (Bio-Rad). Control lanes labeled ‘ds’ contain an equivalent amount of DNA as reaction lanes but no enzyme. To mimic DNA processing product intensity control lanes labeled ‘ss’ contained half the amount of DNA heat denatured (95°C and gradually cooled to room temperature) to mimic ssDNA products that would be produced as a result of DNA end processing (31 (link)).

Hartenian E., Mendez A.S., Didychuk A.L., Khosla S, & Glaunsinger B.A. (2022). DNA processing by the Kaposi's sarcoma-associated herpesvirus alkaline exonuclease SOX contributes to viral gene expression and infectious virion production. Nucleic Acids Research, 51(1), 182-197.

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Agarose Gel Electrophoresis of mRNA Samples

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A 2% (w/v) of agarose with 0.5 × TBE buffer (45 mM Tris–borate and 1 mM EDTA) and 5.5 mM of magnesium chloride was prepared and pre-stained with Invitrogen SYBR^® Safe DNA Gel Stain (1:10,000 dilution). The gel was loaded with 1.5 μL of mRNA sample diluted in WFI into a final volume of 10 μL and 2 μL of 6 × purple Loading Dye (New England Biolabs, UK). A 5 μL of 1 kb Plus DNA Ladder (New England Biolabs, UK) was used as the molecular marker. The electrophoresis was run at 100 V for 75 min using 0.5 × TBE buffer containing 5.5 mM of MgCl₂. The gel was visualised using Amersham™ Imager 600 (GE Healthcare, UK).

Sari Y., Sousa Rosa S., Jeffries J, & Marques M.P. (2024). Comprehensive evaluation of T7 promoter for enhanced yield and quality in mRNA production. Scientific Reports, 14, 9655.

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DNA Binding Assay of Purified Toxin

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Dilutions of purified toxin (0–16 µM) were incubated with mRNA (100 ng), pGEM-TEasy plasmid (50 ng) and genomic DNA from H. pylori strain P12 (100 ng) in reaction buffer (20 mM Tris–HCl pH 7.0, 50 mM NaCl, 2.5 mM MgSO₄) for 30 min at 37 °C. Reactions were quenched by addition of 6 × purple loading dye (New England BioLabs) then analysed by electrophoresis on 0.8% agarose gels stained with ethidium bromide. Purified NcoI-restricted linear plasmid DNA (30 ng) was optionally included as a size marker. Reaction buffer was also prepared with 2.5 mM of each of MgCl₂, MnCl₂, CuCl₂, ZnCl₂, NiCl₂ and CaCl₂ as required.
Relevant templates were prepared using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich), Nucleospin Plasmid DNA purification kit (Macherey–Nagel) or RNeasy Plus Mini kit (Qiagen) as appropriate. Messenger RNA was transcribed in vitro from PCR-amplified recA or tfiT template using the HiScribe T7 Quick High Yield RNA Synthesis kit (New England BioLabs). The quality and concentration of DNA and RNA samples was monitored with a Nanodrop spectrophotometer (Thermo Scientific).

Boampong K., Smith S.L, & Delahay R.M. (2020). Rapid growth inhibitory activity of a YafQ-family endonuclease toxin of the Helicobacter pylori tfs4 integrative and conjugative element. Scientific Reports, 10, 18171.

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Detecting Ribonucleotides in DNA

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Ribonucleotides in DNA are visualized using alkaline-agarose electrophoresis. Treatment with NaOH fragments DNA 3′ to an embedded rNMP, resulting in DNA that migrates faster by alkaline-agarose electrophoresis. 2 m NaOH (or 5 m NaCl as negative control) was added to DNA at 0.3 m final concentration and heated at 55 °C for 2 h to hydrolyze at ribonucleotide positions in DNA. 6× Purple loading dye (New England Biolabs, Inc.) was added to samples before loading onto a 1% alkaline-agarose gel (1% agarose, 1 mm EDTA, 50 mm NaOH). The gel was run at 5 V for 16 h, stained with SYBR Gold (Thermo Fisher Scientific), and visualized on a UV imager.

Heider M.R., Burkhart B.W., Santangelo T.J, & Gardner A.F. (2017). Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea. The Journal of Biological Chemistry, 292(21), 8835-8845.

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γPNA Invasion Assay for dsDNA

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γPNA
invasion was carried out in 10 μL reaction volume including
1× MOPS buffer (20 mM MOPS pH 7.0, 5 mM CH₃COONa,
1 mM EDTA). 2 μM γPNA molecules were treated with 20 nM
of specific linear dsDNA target (609 bp) and incubated at 37 °C
for 6 h. The dsDNA target was produced by PCR amplification of the
pUC19 target containing PNA-binding regions, using 1447 forward and
1448 reverse primers listed in Table S1. The γPNA-invaded dsDNA reactions and the noninvaded dsDNA
templates were separately mixed with 6× purple loading dye (NEB,
B7024S) and run on a 6% native TBE gel at 120 V for 1 h 15 min. The
gel was subsequently stained with 1× TBE buffer containing 1×
SYBR Gold (Invitrogen, S11494) for <10 min and imagined in an iBright
1500 imaging system (Thermo Scientific, A44114).

Sivakrishna Rao G., Saleh A.H., Melliti F., Muntjeeb S, & Mahfouz M. (2024). Harnessing Peptide Nucleic Acids and the Eukaryotic Resolvase MOC1 for Programmable, Precise Generation of Double-Strand DNA Breaks. Analytical Chemistry, 96(6), 2599-2609.

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