Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *High Sensitivity*
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H340|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R68|
|Component A: Helixyte Green™||1 vial (1 mL, 200X in DMSO)|
|Component B: 20X Assay Buffer||1 bottle (25 mL)|
|Component C: Calf thymus DNA Standard||1 vial (1 mL, 100 µg/mL)|
AT A GLANCE
- Add 1mL dsDNA standards or test samples to each cuvette
- Add 1mL Helixyte Green™ working solution
- Incubate at RT for 5-10 minutes
- Monitor the fluorescence at Ex/Em=490/525 nm
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 is 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.
PREPARATION OF STOCK SOLUTION
1. Assay Buffer (1X)
Prepare a 1X Assay buffer by diluting the concentrated buffer 20-fold with sterile, distilled, DNase-free water.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/17651
For high range standard curve:
Add 30 µL of 100 µg/mL dsDNA stock solution (Component C) to 1.47 mL of 1X Assay buffer to have 2000 ng/mL dsDNA solution, and then perform 1:2 and 1:10 serial dilutions to get 1000, 100, 10, 1 and 0 ng/mL.
For low range standard curve:
Add 40 µL of 2 µg/mL dsDNA stock solution to 1.56 mL of 1X Assay buffer to have 50 ng/mL dsDNA solution, and then perform 1:2 and 1:10 serial dilutions to get 25, 2.5, 0.25, 0.025 and 0 ng/mL.
PREPARATION OF WORKING SOLUTION
Prepare Helixyte Green™ working solution by making 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 2 mL final volume, 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 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.
SAMPLE EXPERIMENTAL PROTOCOL
- Add 1 mL of Helixyte Green™ working solution to each cuvette containing 1 mL of the dsDNA standard, blank control, and test samples to make the total dsDNA assay volume of 2 mL/cuvette.
- Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.
- Monitor the fluorescence increase with a spectroflurometer at Ex/Em = 490/525 nm. Note: To minimize photobleaching effects, keep the time for fluorescence measurement constant for all samples.
Open in Advanced Spectrum Viewer
|Name||Excitation (nm)||Emission (nm)|
|Helixyte™ Green Fluorimetric ssDNA Quantitation Kit *Optimized for Microplate Readers*||498||519|
Authors: Peterson EJ, Kireev D, Moon AF, Midon M, Janzen WP, Pingoud A, Pedersen LC, Singleton SF.
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