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Helixyte™ Green ssDNA reagent

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.

Example protocol

AT A GLANCE

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 

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/17620


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).

PREPARATION OF WORKING SOLUTION

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. 

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
BL BL TS TS
SS1 SS1
SS2 SS2
SS3 SS3    
SS4 SS4    
SS5 SS5    
SS6 SS6    
SS7 SS7    
Table 2.The reagent composition for each well.
WellVolumeReagent
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). 

Spectrum

References

View all 50 references: Citation Explorer
Assessment of AMBER Force Fields for Simulations of ssDNA.
Authors: Oweida, Thomas J and Kim, Ho Shin and Donald, Johnny M and Singh, Abhishek and Yingling, Yaroslava G
Journal: Journal of chemical theory and computation (2021): 1208-1217
One-Pot Synthesis of Defined-Length ssDNA for Multiscaffold DNA Origami.
Authors: Noteborn, Willem E M and Abendstein, Leoni and Sharp, Thomas H
Journal: Bioconjugate chemistry (2021): 94-98
RPA2 winged-helix domain facilitates UNG-mediated removal of uracil from ssDNA; implications for repair of mutagenic uracil at the replication fork.
Authors: Kavli, Bodil and Iveland, Tobias S and Buchinger, Edith and Hagen, Lars and Liabakk, Nina B and Aas, Per A and Obermann, Tobias S and Aachmann, Finn L and Slupphaug, Geir
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
Page updated on October 4, 2024

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Physical properties

Solvent

DMSO

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

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501

Platform

Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black