Portelite™ Fluorimetric DNA Quantitation Kit with Broad Dynamic Range *Optimized for Cytocite™ and Qubit™ Fluorometers*

Protocol summary

1. Prepare Helixyte™ Green BR working solution
2. Add 190 uL 1X Helixyte™ Green BR working solution into each 0.2 mL PCR tube
3. Add 10 uL DNA standards or test samples into each tube
4. Incubate at room temperature for 2 minutes
5. Monitor fluorescence with CytoCite™ fluorometer or Qubit™

Important notes
Bring all the components at room temperature before opening.

Helixyte™ Green BR working solution:
Make a 200-fold dilution of Portelite™ dsDNA reagent (Component A) in DNA Assay Buffer (Component B). For example, to prepare enough working solution for 8 samples, add 5 uL of Helixyte™ Green BR (Component A) into 1 mL of DNA Assay Buffer (Component B). Protect the working solution from light by covering it with foil or placing it in the dark. Note: Protect the working solution from light by covering it with foil or placing it in the dark. 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.

The acceptable sample volume could be a range from 1~20 uL depending on the estimate concentration of DNA sample. The recommend sample volume is 10 uL with the DNA concentration in 0.2~100 ng/uL range. If other sample volume is being used, please adjust the dilution factor in the concentration calculations. 

 The following protocol is generated based on 10 uL sample volume with the DNA concentration in 0.2~100 ng/uL range.

1. Add 190 uL 1X Helixyte™ Green BR working solution into each Cytocite™ sample tube (#CCT100) or equivalent 0.2 mL PCR tube. Note: Use thin-wall, polypropylene, clear 0.2 mL PCR tubes such as#CCT100.
   
   
2. Add DNA standards or test samples 10 uL into each tube, and then mix by vortexing 2~3 seconds.
   
   
3. Incubate all tubes at room temperature for 2 minutes.
   
   
4. Insert the samples into CytoCite™ or Quibit™ and monitor the fluorescence with green fluorescence channel. Follow the procedure appropriate for CytoCite™ Fluorometer. See the link below for detailed instructions: https://devices.aatbio.com/documentation/user-manual-for-cytocite-fluorometer

PREPARATION OF STANDARD CALIBRATION CURVE

For Portelite™ assays, you have the choice to make a calibration curve with the DNA standards. Here is a brief protocol to generate a customized DNA standard curve:

1. Perform 1/3 serial dilution with 100 ng/uL with DNA Standard BR #2 (Component D) in DNA Assay Buffer (Component B) to get 30, 10, 3, 1, 0.3, 0.1 and 0 ng/uL DNA standard dilutions.
   
   
2. Add 190 uL of Helixyte™ Green BR working solution into each tube.
   
   
3. Add 10 uL standards or 10 uL samples into a 0.2 mL PCR tube.
   
   
4. Incubate the reaction at room temperature for 2 minutes.
   
   
5. Insert the samples into CytoCite™ and monitor the fluorescence with green fluorescence channel.