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

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

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.

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

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

Table 2. The reagent composition for each well.

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).