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Helixyte™ Green Fluorimetric ssDNA Quantitation Kit *Optimized for Microplate Readers*
Helixyte™ Green Fluorimetric ssDNA Quantitation Kit is designed to measure single-stranded DNA in an easy and accurate format. The kit has all the essential reagents, including Helixyte™ Green ssDNA reagent, dilution buffer, and prediluted DNA standards. Helixyte™ Green ssDNA reagent is a sensitive fluorescent nucleic acid probe for quantifying oligonucleotides and single-stranded DNA (ssDNA) in solution. It enables researchers to quantify as little as 100 pg/mL oligonucleotide or ssDNA. This sensitivity exceeds that achieved with absorbance methods by more than 10,000-fold. With this kit, as little as 1 ng/mL oligonucleotide or ssDNA might be detected. A few ssDNAs were quantified with this kit, including M13 and ϕX174 viral DNA and denatured calf thymus DNA that had similar sensitivity. Their detection limits were not significantly interfered by the common contaminants in nucleic acid preparations, including salts, urea, ethanol, chloroform, detergents, proteins, ATP, nucleotides and short oligonucleotides of six bases. However, double-stranded DNA (dsDNA) and RNA do interfere with the assay as Helixyte™ Green ssDNA reagent binds to dsDNA and RNA to generate additional fluorescence signals. Helixyte™ Green Fluorimetric ssDNA Quantitation Kit is optimized for quantifying ssDNA with a fluorescence microplate reader.
Example protocol
AT A GLANCE
Protocol summary
- Add 100 µL of ssDNA Standards or test samples
- Add 100 µL of Helixyte™ Green ssDNA working solution
- Incubate at RT for 5-10 minutes
- Monitor the fluorescence intensity at Ex/Em=490/525 nm
Important
The following protocol is an example of quantifying the ssDNA using Helixyte™ Green ssDNA. Allow all the components to warm to room temperature before opening. No data are available on the mutagenicity or toxicity of Helixyte™ Green ssDNA stain. Because this reagent binds to nucleic acids, it should be treated as a potential mutagen and handled with appropriate care. The DMSO stock solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues.PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/
https://www.aatbio.com/tools/serial-dilution/
ssDNA Standard solution
Add 10 uL of 100 ug/mL ssDNA Standard solution (Component C) to 190 uL of Assay Buffer (Component B) to get a 5 ug/mL standards solution, then perform 1:3 dilutions to obtain serially diluted ssDNA standards (SS2-SS7).PREPARATION OF WORKING SOLUTION
Helixyte™ Green ssDNA working solution
Prepare the Helixyte™ Green ssDNA working solution by adding 100 μL of Helixyte™ Green ssDNA (Component A) into 10 mL of Assay Buffer (Component B). Protect the working solution from light by covering it with foil or placing it in the dark.Note It’s recommended to prepare the working solution in a plastic container rather than a glass container, as the dye may adsorb to the glass surface. For best results, this solution should be used within a few hours after the dilution.
Note 10 mL of working solution is enough for one 96-well plate.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1.The layout of ssDNA Standards and test samples in a solid black 96-well microplate. SS= ssDNA Standards (SS1 - SS7, 1667 to 2.3 ng/mL); BL=Blank Control; TS=Test Samples
Table 2.The reagent composition for each well.
| BL | BL | TS | TS |
| SS1 | SS1 | … | … |
| SS2 | SS2 | … | … |
| SS3 | SS3 | ||
| SS4 | SS4 | ||
| SS5 | SS5 | ||
| SS6 | SS6 | ||
| SS7 | SS7 |
| Well | Volume | Reagent |
| SS1-SS7 | 100 µL | Serial dilutions ( 1667 to 2.3 ng/mL) |
| BL | 100 µL | Assay Buffer |
| TS | 100 µL | Sample |
- Prepare ssDNA Standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 100 µL.
- Add 100 µL of the Helixyte™ Green ssDNA working solution to each well of ssDNA Standards, blank control, and test samples to make the assay volume of 200 µL/well. For a 384-well plate, add 25 µL of the Helixyte™ Green ssDNA working solution into each well instead, to get a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm (cut off at 515 nm).
Spectrum
Product family
References
View all 24 references: Citation Explorer
An OliGreen-responsive fluorescence sensor for sensitive detection of organophosphorus pesticide based on its specific selectivity towards T-Hg2+-T DNA structure.
Authors: Zhou, Xiaoyuan and Wang, Chenchen and Wu, Lina and Wei, Wei and Liu, Songqin
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2021): 119155
Authors: Zhou, Xiaoyuan and Wang, Chenchen and Wu, Lina and Wei, Wei and Liu, Songqin
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2021): 119155
A novel detection of radon based on its decay product inducing conformational changes of an aptamer probe.
Authors: Long, Minzhi and Deng, Han and Tian, Gang and Song, Chunli and Liu, Hongwen and Shen, Yi and Lv, Changyin
Journal: Analytica chimica acta (2016): 202-7
Authors: Long, Minzhi and Deng, Han and Tian, Gang and Song, Chunli and Liu, Hongwen and Shen, Yi and Lv, Changyin
Journal: Analytica chimica acta (2016): 202-7
Studies of DNA Aptamer OliGreen and PicoGreen Fluorescence Interactions in Buffer and Serum.
Authors: Bruno, John G and Sivils, Jeffrey C
Journal: Journal of fluorescence (2016): 1479-87
Authors: Bruno, John G and Sivils, Jeffrey C
Journal: Journal of fluorescence (2016): 1479-87
Accumulation of single-stranded DNA in Escherichia coli carrying the colicin plasmid pColE3-CA38.
Authors: Morales, Magali and Attai, Hedieh and Troy, Kimberly and Bermudes, David
Journal: Plasmid (2015): 7-16
Authors: Morales, Magali and Attai, Hedieh and Troy, Kimberly and Bermudes, David
Journal: Plasmid (2015): 7-16
Probing the inherent stability of siRNA immobilized on nanoparticle constructs.
Authors: Barnaby, Stacey N and Lee, Andrew and Mirkin, Chad A
Journal: Proceedings of the National Academy of Sciences of the United States of America (2014): 9739-44
Authors: Barnaby, Stacey N and Lee, Andrew and Mirkin, Chad A
Journal: Proceedings of the National Academy of Sciences of the United States of America (2014): 9739-44
Page updated on November 21, 2024
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