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Helixyte™ Green Fluorimetric Total Nucleic Acid Quantitation Kit *Optimized for Microplate Readers*

OverviewpdfSDSpdfProtocol


Excitation (nm)
509
Emission (nm)
529
Helixyte™ Green Fluorimetric Total Nucleic Acid Quantitation Kit is designed to measure total amounts of nucleic acids, including double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) and RNA in an easy and accurate format. The kit has all the essential reagents, including Helixyte™ Green All reagent, dilution buffer, and pre-diluted DNA standards. Helixyte™ Green All reagent is a sensitive fluorescent nucleic acid probe for measuring the total amounts of nucleic acids in a sample that may contain dsDNA, ssDNA, RNA and long oligonucleotides. Helixyte™ Green All reagent indiscriminately binds to dsDNA, ssDNA and RNA. Helixyte™ Green Fluorimetric Total Nucleic Acid Quantitation Kit is optimized for measuring the total amounts of nucleic acids with a fluorescence microplate reader.

Platform


Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Components


Component A: Helixyte™ Green All1 vial (200 µL)
Component B: Assay Buffer1 bottle (50 mL)
Component C: Nucleic Acid Standard200 µL (100 µg/mL)

Example protocol


AT A GLANCE

Protocol Summary
  1. Add 100 µL of Nucleic Acid Standards or test samples
  2. Add 100 µL of Helixyte™ Green All working solution
  3. Incubate at room temperature 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 total nucleic acid content using Helixyte™ Green All. Allow all the components to warm to room temperature before opening. No data are available on the mutagenicity or toxicity of Helixyte™ Green All, the total nucleic acid 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/


Nucleic Acid Standard
Add 10 uL of 100 ug/mL Nucleic Acid Standard solution (Component C) to 190 uL of Assay Buffer (Component B) to get a 5 ug/mL standard solution and then perform 1:3 dilutions to obtain serially diluted Nucleic Acid Standard (NS1-NS7).

PREPARATION OF WORKING SOLUTION

Helixyte™ Green All working solution
Prepare the Helixyte™ Green All working solution by adding 100 μL of Helixyte™ Green All (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 the working solution is enough for one 96-well plate.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1.The layout of Nucleic Acid Standards and test samples in a 96-well solid black microplate. NS= Nucleic Acid Standards (NS1 - NS7, 1667 to 2.3 ng/mL); BL=Blank Control; TS=Test Samples
BL BL TS TS
NS1 NS1
NS2 NS2
NS3 NS3    
NS4 NS4    
NS5 NS5    
NS6 NS6    
NS7 NS7    
Table 2.The reagent composition for each well.
Well Volume Reagent
NS1-NS7 100 µL Serial dilutions ( 1667 to 2.3 ng/mL)
BL 100 µL Assay Buffer
TS 100 µL Sample
  1. Prepare Nucleic Acid 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 All working solution to each well of Nucleic Acid 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 All 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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)509
Emission (nm)529

References


View all 50 references: Citation Explorer
Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings.
Authors: Coole, Jackson and Kortum, Alex and Tang, Yubo and Vohra, Imran and Maker, Yajur and Kundrod, Kathryn and Natoli, Mary and Richards-Kortum, Rebecca
Journal: Journal of visualized experiments : JoVE (2021)
Quantifying viscosity and surface tension of multicomponent protein-nucleic acid condensates.
Authors: Alshareedah, Ibraheem and Thurston, George M and Banerjee, Priya R
Journal: Biophysical journal (2021): 1161-1169
High-resolution visualization and quantification of nucleic acid-based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS).
Authors: He, Cuiwen and Migawa, Michael T and Chen, Kai and Weston, Thomas A and Tanowitz, Michael and Song, Wenxin and Guagliardo, Paul and Iyer, K Swaminathan and Bennett, C Frank and Fong, Loren G and Seth, Punit P and Young, Stephen G and Jiang, Haibo
Journal: Nucleic acids research (2021): 1-14
Nodal metastatic load in papillary thyroid carcinoma. Morphological and molecular analysis with one-step nucleic acid amplification on more than 550 lymph nodes.
Authors: Iglesias, Carmela and González, Oscar and Temprana-Salvador, Jordi and García-Burillo, Amparo and Caubet, Enric and Ramón Y Cajal, Santiago and Zafon, Carles
Journal: Endocrinologia, diabetes y nutricion (2021): 346-353
Role of highly sensitive nucleic acid amplification testing for plasma cytomegalovirus DNA load in diagnosis of cytomegalovirus gastrointestinal disease among kidney transplant recipients.
Authors: Taksinwarajarn, Touchapong and Sobhonslidsuk, Abhasnee and Kantachuvesiri, Surasak and Thongprayoon, Charat and Cheungpasitporn, Wisit and Bruminhent, Jackrapong and ,
Journal: Transplant infectious disease : an official journal of the Transplantation Society (2021): e13635
High throughput quantification of short nucleic acid samples by capillary electrophoresis with automated data processing.
Authors: Dangerfield, Tyler L and Huang, Nathan Z and Johnson, Kenneth A
Journal: Analytical biochemistry (2021): 114239
Assessing Nucleic Acid: Cationic Nanoparticle Interaction for Gene Delivery.
Authors: Singh, Moganavelli
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 43-55
Integrated microneedle-smartphone nucleic acid amplification platform for in-field diagnosis of plant diseases.
Authors: Paul, Rajesh and Ostermann, Emily and Chen, Yuting and Saville, Amanda C and Yang, Yuming and Gu, Zhen and Whitfield, Anna E and Ristaino, Jean B and Wei, Qingshan
Journal: Biosensors & bioelectronics (2021): 113312
Is DAPI assay of cellular nucleic acid reliable in the presence of protein aggregates?
Authors: Mora, Aruna K and Khan, Sufiyan and Patro, Birija S and Nath, Sukhendu
Journal: Chemical communications (Cambridge, England) (2020): 13844-13847
Rapid, Cost-Effective Peptide/Nucleic Acid-Based Platform for Therapeutic Antibody Monitoring in Clinical Samples.
Authors: Mocenigo, Marco and Porchetta, Alessandro and Rossetti, Marianna and Brass, Erik and Tonini, Lucia and Puzzi, Luca and Tagliabue, Elda and Triulzi, Tiziana and Marini, Bruna and Ricci, Francesco and Ippodrino, Rudy
Journal: ACS sensors (2020): 3109-3115