Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *Optimized for Broad Dynamic Range*

1. Prepare dSDNA standards, test samples and dye working solution
2. Add DNA standards or test samples (50 uL)
3. Add Helixyte™ Green BR working solution (50 uL)
4. Incubate at room temperature for 2 minutes
5. Monitor fluorescence intensity at Ex/Em= 490/530 nm

The following protocol is provided as an example for quantifying dsDNA with Helixyte™ Green BR. Warm all the components to room temperature before opening. No data are available for the mutagenicity or toxicity of Helixyte™ Green dsDNA 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.

Add 50 uL Helixyte™ Green BR (Component A) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.050 mL. Protect the working solution from light by covering it with foil or placing it in the dark.

Note: We recommend preparing this solution in a plastic container rather than glass, as the dye may adsorb to glass surfaces. For best results, this solution should be used within a few hours of its preparation.

Table 1. Layout of dsDNA standards and test samples in a clear bottom 96-well microplate. DS=dsDNA standards (DS1-DS7, 20 to 0.027 ug/mL); BL=Blank Control; TS=Test Samples

Table 2. Reagent composition for each well.

1. Prepare dsDNA standards (DS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 uL of reagent per well instead of 50 uL.

   Note: Treat cells or tissue samples as desired.

2. Add 50 uL of Helixyte™ Green BR dye working solution (2X) to each well of dsDNA standard, blank control, and test samples to make the total assay volume 100 uL/well. For a 384-well plate, add 25 uL ofHelixyte™ Green BR dye working solution into each well instead, for a total volume of 50 uL/well.
3. Incubate the reaction for 2 minutes at room temperature, protected from light.
4. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 490/530 nm (cut off at 515nm).