This protocol only provides a guideline, and should be modified according to your specific needs.Protocol summary
- Prepare Helixyte™ Green working solution
- Add 190 µL 1X Helixyte Green™ working solution into each 0.2 mL PCR tube (Cat#: CCT100)
- Add 10 µL DNA standards or test samples into each tube
- Incubate at room temperature for 2 minutes
- Monitor fluorescence with CytoCite™ fluorometer or Qubit™ fluorometer
Important notes
Bring all the kit components at room temperature before starting the experiment.
| Instrument: | CytoCite Fluorometer |
| Excitation: | 480 nm |
| Emission: | 520 nm |
| Instrument specification(s): | 0.2 mL, thin-wall PCR tube |
| | |
| Instrument: | Qubit Fluorometer |
| Excitation: | 480 nm |
| Emission: | 520 nm |
| Instrument specification(s): | 0.2 mL, thin-wall PCR tube |
Helixyte Green™ working solution:
To prepare enough working solution for 10 samples, add 10 μL of Helixyte Green™ (Component A) into 2 mL of DNA Assay Buffer (Coponent B). Note: 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.
The acceptable sample volume could be a range from 1~20 µL depending on the estimate concentration of DNA sample. The recommend sample volume is 10 µL with the DNA concentration in 0.5~10 ng/ µL 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 µL sample volume with the DNA concentration in 0.5~10 ng/µL range.
- Add 190 µL 1X Helixyte Green™ 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.
- Add DNA standards or test samples 10 µL into each tube, and then mix by vortexing 2~3 seconds.
- Allow all tubes to incubate at room temperature for 2 minutes.
- Insert the samples into CytoCite™ or Quibit™ and monitor the fluorescence with green fluorescence channel. Follow the procedure appropriate for CytoCite™ Fluorometer. Note: 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:
- Perform dilution with DNA Assay Buffer to get 10, 8, 6, 4, 2, 1, 0.5, 0 ng/µL DNA standard dilutions.
- Add 190 µL of Helixyte Green™ working solution into a 0.2 mL PCR tube.
- Add 10 µL standards or 10 µL samples into each tube.
- Incubate the reaction at room temperature for 2 minutes.
- Insert the samples into CytoCite™ and monitor the fluorescence with green fluorescence channel.
The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Concentration samples. We recommend using the Online Linear Regression Calculator which can be found at:
https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator
Figure 1. DNA standard curve generated using Portelite™ Fluorimetric DNA High Sensitivity Quantitation Kit. Fluorescence intensity was quantified using FITC channel, regression model was calculated using log-log best-fit. Detection limit was 10 pg/µL.
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.
