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Helixyte™ Green ssDNA reagent

The ssDNA dose response was measured with Helixyte™ Green ssDNA reagent in a 96-well solid black plate.
The ssDNA dose response was measured with Helixyte™ Green ssDNA reagent in a 96-well solid black plate.
The ssDNA dose response was measured with Helixyte™ Green ssDNA reagent in a 96-well solid black plate.
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Physical properties
Spectral properties
Excitation (nm)498
Emission (nm)519
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Excitation (nm)
Emission (nm)
Synthetic oligonucleotides are used in several molecular biology techniques, such as DNA sequencing, site-directed mutagenesis, DNA amplification, and in situ hybridization. The most commonly used technique for measuring oligonucleotide and single-stranded DNA (ssDNA) concentration is the determination of absorbance at 260 nm (A260). However, absorbance methods suffer significant interferences resulting from various contaminants commonly found in nucleic acid preparations, including nucleotides, double-stranded nucleic acids, and proteins. Helixyte™ Green ssDNA Quantifying Reagent is an excellent alternative for quantifying ssDNA and oligonucleotides with significantly improved sensitivity and selectivity. It is a positively charged fluorescent probe that binds to the hydrophobic pockets of ssDNA and forms a highly luminescent complex through the synergistic actions of stacking, hydrophobic forces, hydrogen bonding, and electrostatic interactions. Helixyte™ Green ssDNA reagent has extremely low inherent fluorescence that is significantly enhanced upon binding to ssDNA. The increased sensitivity of Helixyte™ Green ssDNA over absorbance at 260 nm enables researchers to quantify as little as 100 pg/mL oligonucleotide or ssDNA (~500 pg/mL) with a standard spectrofluorometer and fluorescein excitation and emission wavelengths. As little as 1 ng/mL oligonucleotide or ssDNA can be detected with a fluorescence microplate reader. Helixyte™ Green ssDNA reagent has a wide linear range of detection ranging from 100 pg/mL to 1 μg/mL.


Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Example protocol


Protocol summary
  1. Add 100 µL of ssDNA Standards or test samples
  2. Add 100 µL of Helixyte™ Green ssDNA working solution
  3. Incubate at RT for 5-10 minutes
  4. Monitor the fluorescence intensity at Ex/Em=490/525 nm 

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.


For convenience, use the Serial Dilution Planner:

ssDNA Standard solution
Add 10 uL of 100 ug/mL ssDNA Standard solution (Not provided) to 190 uL of buffer of your choice to get a 5 ug/mL standards solution, then perform 1:3 dilutions to obtain serially diluted ssDNA standards (SS2-SS7).


Helixyte™ Green ssDNA working solution
Prepare the Helixyte™ Green ssDNA working solution by adding 100 μL of Helixyte™ Green ssDNA reagent into 10 mL of buffer of your choice. 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.
Note     10 mM Tris-HCl (pH 8.0) with 0.1 mM EDTA can be used to make working solution and standards. 


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
SS3 SS3    
SS4 SS4    
SS5 SS5    
SS6 SS6    
SS7 SS7    
Table 2.The reagent composition for each well.
SS1-SS7100 µLSerial dilutions (1667 to 2.3 ng/mL)
BL100 µLBuffer of your choice
TS100 µLSample
  1. 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.
  2. 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.
  3. Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.
  4. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm (cut off at 515 nm). 


Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)498
Emission (nm)519



View all 50 references: Citation Explorer
Assessment of AMBER Force Fields for Simulations of ssDNA.
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One-Pot Synthesis of Defined-Length ssDNA for Multiscaffold DNA Origami.
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Journal: Nucleic acids research (2021): 3948-3966
Detection of nucleotides in hydrated ssDNA via 2D h-BN nanopore with ionic-liquid/salt-water interface.
Authors: Lee, Jung Soo and Oviedo, Juan Pablo and Bandara, Yapa Mudiyanselage Nuwan Dhananjaya Yapa and Peng, Xin and Xia, Longsheng and Wang, Qingxiao and Garcia, Kevin and Wang, Jinguo and Kim, Min Jun and Kim, Moon Jae
Journal: Electrophoresis (2021): 991-1002
Novel colorimetric aptasensor based on unmodified gold nanoparticle and ssDNA for rapid and sensitive detection of T-2 toxin.
Authors: Zhang, Wenwei and Wang, Yanling and Nan, Mina and Li, Yongcai and Yun, Jianmin and Wang, Yi and Bi, Yang
Journal: Food chemistry (2021): 129128
New DNA-hydrolyzing DNAs isolated from an ssDNA library carrying a terminal hybridization stem.
Authors: Zhang, Canyu and Li, Qingting and Xu, Tianbin and Li, Wei and He, Yungang and Gu, Hongzhou
Journal: Nucleic acids research (2021)
Improving the detection limit of Salmonella colorimetry using long ssDNA of asymmetric-PCR and non-functionalized AuNPs.
Authors: Wang, Lijun and Wu, Xue and Hu, Haixia and Huang, Yukun and Yang, Xiao and Wang, Qin and Chen, Xianggui
Journal: Analytical biochemistry (2021): 114229
Rapid, ultrasensitive and non-enzyme electrochemiluminescence detection of hydrogen peroxide in food based on the ssDNA/g-C3N4 nanosheets hybrid.
Authors: Liu, Zhijun and Wang, Li and Liu, Pengfei and Zhao, Kairen and Ye, Shuying and Liang, Guoxi
Journal: Food chemistry (2021): 129753
Label-free detection of exosomes based on ssDNA-modulated oxidase-mimicking activity of CuCo2O4 nanorods.
Authors: Zhang, Yingzhi and Su, Qiwen and Song, Dan and Fan, Jiayuan and Xu, Zhangrun
Journal: Analytica chimica acta (2021): 9-16
R-loop-Mediated ssDNA Breaks Accumulate Following Short-Term Exposure to the HDAC Inhibitor Romidepsin.
Authors: Safari, Maryam and Litman, Thomas and Robey, Robert and Aguilera, Andres and Chakraborty, Arup R and Reinhold, William and Basseville, Agnes and Petrukhin, Lubov and Scotto, Luigi and O'Connor, Owen A and Pommier, Yves and Fojo, Antonio T and Bates, Susan E
Journal: Molecular cancer research : MCR (2021)