Portelite™ Fluorimetric Total Nucleic Acid Quantitation Kit Optimized for Cytocite™ and Qubit™ Fluorometers

Portelite™ Fluorimetric Total Nucleic Acid Quantitation Kit is designed to rapidly measure the total amounts of nucleic acids, including double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) and RNA in a sample. The kit has all the essential reagents, including Helixyte™ Green ssDNA reagent, dilution buffer, and prediluted 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 double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA and long oligonucleotides. Helixyte™ Green All reagent indiscriminately binds to dsDNA, ssDNA and RNA. Portelite™ Fluorimetric Total Nucleic Acid Quantitation Kit is optimized for measuring the total amounts of nucleic acids with CytoCite™ or Qubit® fluorometers.

Example protocol

AT A GLANCE

Protocol Summary

Prepare a Helixyte™ Green All working solution
Add 190 µL of 1X Helixyte™ Green All working solution into each 0.2 mL PCR tube
Add 10 µL of Nucleic Acid Standards or test samples into each tube
Incubate at room temperature for 2 minutes
Monitor the fluorescence intensity with CytoCite™ fluorometer or Qubit™ fluorometer

Important

All kit components must be brought to room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Helixyte™ Green All working solution

Make a 200-fold dilution of Helixyte™ Green All reagent (Component A) with Assay Buffer (Component B). For example, to prepare enough working solution for 5 samples, add 5 μL of Helixyte™ Green All (Component A) into 1 mL of Assay Buffer (Component B).
Note Protect the working solution from light by covering it with foil or placing it in the dark. It’s recommended to prepare the 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.

SAMPLE EXPERIMENTAL PROTOCOL

The acceptable range for the sample volume could be 1~20 μL depending on the estimated concentration of the Nucleic Acid sample.
The following protocol is generated based on a sample volume of 10 μL.

Add 190 µL of 1X Helixyte™ Green All working solution into each Cytocite™ sample tube (#CCT100) or the equivalent 0.2 mL PCR tube.
Note Use thin-wall, polypropylene, clear 0.2 mL PCR tubes such as AAT Cat#CCT100.
Add 10 μL of Nucleic Acid Standards or test samples into each tube, and then mix by vortexing for 2~3 seconds.
Incubate all tubes at room temperature for 2 minutes.
Insert the samples into CytoCite™ or Qubit™ and monitor the fluorescence intensity with the green fluorescence channel. Follow the appropriate procedures for CytoCite™ Fluorometer. See the link below for detailed instructions: https://devices.aatbio.com/documentation/user-manual-for-cytocite-fluorometer

Preparation of Standard Calibration Curve

For Portelite™ assays, you have the choice to make a calibration curve with the Nucleic Acid Standards. Here is a brief protocol to generate a customized DNA standard curve.

Perform a 1:2 serial dilution: Add 10 ng/μL Nucleic Acid Standard #2 (Component D) into the Assay Buffer (Component B) to get 10, 5, 2.5, 1.25, 0.62, 0.31, 0.15 ng/µL DNA standard dilutions.
Add 190 µL of the Helixyte™ Green All working solution into each tube.
Add 10 µL of standards into a 0.2 mL PCR tube and then mix by vortexing for 2∼3 seconds.
Incubate the reaction at room temperature for 2 minutes.
Insert the samples into CytoCite™ and monitor the fluorescence intensity with the green fluorescence channel.

Spectrum

Open in Advanced Spectrum Viewer

References

View all 13 references: Citation Explorer

Accurate bulk quantitation of droplet digital PCR.
Authors: Sun, Chen and Liu, Leqian and Vasudevan, Harish N and Chang, Kai-Chun and Abate, Adam R
Journal: bioRxiv : the preprint server for biology (2021)

Nucleic Acid Quantitation with Log-Linear Response Hybridization Probe Sets.
Authors: Wu, Lucia R and Fang, John Z and Khodakov, Dmitriy and Zhang, David Yu
Journal: ACS sensors (2020): 1604-1614

Nucleic Acid Extraction from Human Biological Samples.
Authors: Mullegama, Sureni V and Alberti, Michael O and Au, Cora and Li, Yan and Toy, Traci and Tomasian, Vanina and Xian, Rena R
Journal: Methods in molecular biology (Clifton, N.J.) (2019): 359-383

MICROFLUIDIC DEVICES FOR LABEL-FREE AND NON-INSTRUMENTED QUANTITATION OF UNAMPLIFIED NUCLEIC ACIDS BY FLOW DISTANCE MEASUREMENT.
Authors: Chatterjee, Debolina and Mansfield, Danielle S and Woolley, Adam T
Journal: Analytical methods : advancing methods and applications (2014): 8173-8179

Helicase-dependent amplification of nucleic acids.
Authors: Cao, Yun and Kim, Hyun-Jin and Li, Ying and Kong, Huimin and Lemieux, Bertrand
Journal: Current protocols in molecular biology (2013): 15.11.1-15.11.12

Page updated on November 20, 2024

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