Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *Optimized for Microplate Readers*

Protocol summary

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

Important notes
The following protocol is an example for quantifying dsDNA with Helixyte Green™. Allow all the components to warm to room temperature before opening. No data are available addressing 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.

Prepare Helixyte Green™ working solution by adding 50 μL of Helixyte Green™ (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: 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 solid black 96-well microplate. DS= dsDNA Standards (DS1 - DS7, 2.3 to 1667 ng/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 µL of reagent per well instead of 100 µL.
   
   
2. Add 100 µL of Helixyte Green™ working solution to each well of dsDNA standard, blank control, and test samples to make the total dsDNA assay volume of 200 µL/well. For a 384-well plate, add 25 µL of BLANK assay mixture into each well instead, for 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).