Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *High Sensitivity*

1. Add 1mL of dsDNA standards or test samples to each cuvette.

2. Add 1 mL of the Helixyte Green™ working solution.

3. Incubate at room temperature for 5 to 10 minutes.

4. Monitor the fluorescence at Ex/Em = 490/525 nm.

The following protocol is an example for quantifying dsDNA with Helixyte Green™. Allow all components to warm to room temperature before opening. There is no available data on the mutagenicity or toxicity of Helixyte Green™ dsDNA stain. However, since this reagent binds to nucleic acids, it should be treated as a potential mutagen and handled with appropriate care. Handle the DMSO stock solution with particular caution, as DMSO is known to facilitate the entry of organic molecules into tissues.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

1. Prepare a 1X Assay buffer by diluting the concentrated buffer 20-fold with sterile, distilled, DNase-free water.

1. To prepare the Helixyte Green™ working solution, make a 200-fold dilution of the concentrated DMSO solution in 1X assay buffer. For example, to prepare enough working solution to assay 10 samples in a final volume of 2 mL each, add 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 a dark location.

   Note: We recommend using a plastic container to prepare this solution, as the dye can adhere to glass surfaces. For optimal performance, use the solution within a few hours of preparation.

1. Add 1 mL of Helixyte Green™ working solution to each cuvette containing 1 mL of the dsDNA standard, blank control, and test samples. This will bring the total volume of the dsDNA assay in each cuvette to 2 mL.

2. Incubate the reaction at room temperature for 5-10 minutes, keeping it protected from light.

3. Monitor the fluorescence increase with a spectrofluorometer at Ex/Em = 490/525 nm, Cutoff = 515 nm.
   
   Note: To minimize photobleaching effects, keep the time for fluorescence measurement constant for all samples.