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Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution*

Helixyte™ Green is an excellent nucleic acid sensor that exhibits large fluorescence enhancement upon binding to dsDNA. It has the same spectral properties to those of PicoGreen®, thus a great replacement to PicoGreen® (PicoGreen® is the trademark of Invitrogen). The most commonly used technique for measuring nucleic acid concentration is the determination of absorbance at 260 nm (A260). However, the absorbance method suffers great interferences resulted from various contaminants commonly found in nucleic acid preparations, including nucleotides, single-stranded nucleic acids and proteins. Helixyte™ Green dsDNA Quantifying Reagent is an excellent alternative for quantifying DNAs with greatly improved sensitivity and selectivity. Helixyte™ Green dsDNA Quantifying Reagent is an ultra-sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in molecular biological procedures such as cDNA synthesis for library production and DNA fragment purification for subcloning, as well as diagnostic applications, such as quantitating DNA amplification products and primer extension assays. Using the Helixyte™ Green dsDNA Quantifying Reagent, you can selectively detect as little as 25 pg/ml of dsDNA in the presence of ssDNA, RNA, and free nucleotides. The assay is linear over three orders of magnitude and has little sequence dependence, allowing you to accurately measure DNA from many sources, including genomic DNA, viral DNA, miniprep DNA, or PCR amplification products. Helixyte™ Green dsDNA Quantifying Reagent has a few orders of magnitude more sensitive than the UV absorbance readings, saving on precious sample. It is specific for dsDNA in the presence of equimolar amounts of RNA.

Example protocol

PREPARATION OF WORKING SOLUTION

The following example is a protocol for quantifying dsDNA with Helixyte Green™. Before opening, allow the vial of Helixyte Green™ to warm to room temperature.


Note: Exercise caution when working with Helixyte Green™ dsDNA stain, as there is currently no available data regarding its potential mutagenicity or toxicity. Due to its affinity for nucleic acids, this reagent should be handled as a potential mutagen and with appropriate care. Additionally, take special care when handling the DMSO stock solution, as DMSO has been shown to facilitate the entry of organic molecules into tissues.

Preparing the Helixyte Green™ working solution
  1. To prepare an aqueous working solution of Helixyte Green™, dilute the concentrated DMSO solution 200-fold in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5-8.0). For example, adding 50 μL of Helixyte Green™ to 10 mL of TE will prepare enough working solution to assay 100 samples in a final volume of 200 µL. Protect the working solution from light by covering it with foil or placing it in the dark.
     
    Note: We recommend preparing this solution in a plastic container rather than glass, as the dye may adsorb to glass surfaces.
     
    Note:  For best results, this solution should be used within a few hours of its preparation. 

SAMPLE EXPERIMENTAL PROTOCOL

Prepare serial dilutions of dsDNA standard (0 to 3 ng/mL)
  1. Prepare a 1 mg/mL stock solution of dsDNA (such as calf thymus DNA from Sigma) in ddH2O

  2. To prepare a 10 μg/mL dsDNA solution, add 10 μL of the 1 mg/mL dsDNA stock solution (from Step 2.1) to 990 µL of TE buffer. Next, perform 1:10 and 1:2 serial dilutions to obtain concentrations of 1000, 100, 50, 25, 12.5, 6.25, 3.125, and 0 ng/mL.

  3. Add the dsDNA standards and test samples containing DNA into a solid black 96-well microplate, following the instructions provided in Tables 1 and 2.

Table 1. The layout of dsDNA standards and test samples in a solid black 96-well microplate.*

BL
BL

TS

TS

....

....

    

    

    

    

    

    

DS1

DS1

....

....

....

....

    

    

    

    

    

    

DS2

DS2

....

....

....

....

    

    

    

    

    

    

DS3

DS3

    

    

    

    

    

    

DS4

DS4

    

    

    

    

    

    

DS5

DS5

    

    

    

    

    

    

DS6

DS6

    

    

    

    

    

    

DS7

DS7

    

    

    

    

    

    

*Note: DS = dsDNA Standards; BL= Blank Control; TS =Test Samples

Table 2. Reagent composition for each well.* 

dsDNA Standard

Blank Control

Test Sample

Serial dilutions* (100 µL)

TE: 100 μL

100 μL

*Note: Add the serial dilutions of dsDNA standards from 0.1 to 1000 ng/mL into wells from DS1 to DS7 in duplicate. 

Run dsDNA assay
  1. Add 100 μL of dsDNA assay mixture (from Step 1.1) to each well of the dsDNA standard, blank control, and test samples (see Step 2.3) to make the total dsDNA assay volume of 200 µL/well.

    Note: For a 384-well plate, add 25 μL sample and 25 μL of dsDNA assay mixture per well.

    Note: For cuvette-based assays, add 1mL sample and 1mL of dsDNA assay mixture per cuvette.

  2. Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.

  3. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm, cut off at 515 nm.

  4. The fluorescence obtained in the blank wells (containing only TE buffer) is used as a control and will be subtracted from the values obtained in the wells containing dsDNA reactions. The DNA concentrations of the test samples can be determined by referencing the standard curve generated in the DNA Standard Curve step.

Spectrum

Citations

View all 6 citations: Citation Explorer
Modulatory Effects of Isolated Lactobacillus paracasei from Malaysian Water Kefir Grains on the Intestinal Barrier and Gut Microbiota in Diabetic Mice
Authors: Talib, Noorshafadzilah and Mohamad, Nurul Elyani and Ho, Chai Ling and Masarudin, Mas Jaffri and Alitheen, Noorjahan Banu
Journal: Probiotics and Antimicrobial Proteins (2024): 1--13
Arthropods, nematodes, fungi, and bacteria associated with penguin carrion in Barton Peninsula, King George Island, Antarctica
Authors: ZAINI, NUR ADILLA and Low, Van Lun and Gebrelassie, Selam Solomon and Ismail, Siti Sofo and Mahmud, Mohd Hafizi and Houssaini, Jamal and Lee, Won Young and Tomberlin, Jeffery K and Heo, Chong Chin
Journal: (2023)
Bovine Fibroblast-Derived Extracellular Matrix Promotes the Growth and Preserves the Stemness of Bovine Stromal Cells during In Vitro Expansion
Authors: Lee, Kathleen and Jackson, Anisha and John, Nikita and Zhang, Ryan and Ozhava, Derya and Bhatia, Mohit and Mao, Yong
Journal: Journal of Functional Biomaterials (2023): 218
Faecal DNA metabarcoding reveals novel bacterial community patterns of critically endangered Southern River Terrapin, Batagur affinis
Authors: Salleh, Mohd Hairul Mohd and Esa, Yuzine and Ngalimat, Mohamad Syazwan and Chen, Pelf Nyok
Journal: PeerJ (2022): e12970
Archaeal and methanogenic communities in the rice field under different fertilizer applications
Authors: Fatma, Yuli and RUSMANA, IMAN and WAHYUDI, ARIS TRI and others,
Journal: Biodiversitas Journal of Biological Diversity (2019)

References

View all 31 references: Citation Explorer
Inhibitors of Streptococcus pneumoniae surface endonuclease EndA discovered by high-throughput screening using a PicoGreen fluorescence assay
Authors: Peterson EJ, Kireev D, Moon AF, Midon M, Janzen WP, Pingoud A, Pedersen LC, Singleton SF.
Journal: J Biomol Screen (2013): 247
Validation of a PicoGreen-based DNA quantification integrated in an RNA extraction method for two-dimensional and three-dimensional cell cultures
Authors: Chen Y, Sonnaert M, Roberts SJ, Luyten FP, Schrooten J.
Journal: Tissue Eng Part C Methods (2012): 444
Comparison of SYBR Green I-, PicoGreen-, and [3H]-hypoxanthine-based assays for in vitro antimalarial screening of plants from Nigerian ethnomedicine
Authors: Abiodun OO, Gbotosho GO, Ajaiyeoba EO, Happi CT, Hofer S, Wittlin S, Sowunmi A, Brun R, Oduola AM.
Journal: Parasitol Res (2010): 933
Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen dye
Authors: Moreno LA, Cox KL.
Journal: J Vis Exp. (2010)
Metal-enhanced PicoGreen fluorescence: application to fast and ultra-sensitive pg/ml DNA quantitation
Authors: Dragan AI, Bishop ES, Casas-Finet JR, Strouse RJ, Schenerman MA, Geddes CD.
Journal: J Immunol Methods (2010): 95
Page updated on December 6, 2024

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Unit size
1 ml
10 ml
Catalog Number
1759717598
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Physical properties

Molecular weight

661.17

Solvent

DMSO

Spectral properties

Excitation (nm)

502

Emission (nm)

522

Storage, safety and handling

H-phraseH303, H313, H340
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R68

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC41116134

Platform

Fluorescence microplate reader

Excitation490
Emission525
Cutoff515
Recommended plateSolid black
Comparison of calf thymus DNA dose response with Helixyte Green™ (blue circle) and PicoGreen® (red square) in a solid black 96-well microplate using a Gemini fluorescence microplate reader.
Comparison of calf thymus DNA dose response with Helixyte Green™ (blue circle) and PicoGreen® (red square) in a solid black 96-well microplate using a Gemini fluorescence microplate reader.
Comparison of calf thymus DNA dose response with Helixyte Green™ (blue circle) and PicoGreen® (red square) in a solid black 96-well microplate using a Gemini fluorescence microplate reader.