Technical Note 142

Broad Range Assay Standard Protocol

Introduction

The DeNovix® dsDNA Broad Range Assay enables the accurate detection of double-stranded DNA (dsDNA) samples with a standard detection range from 2 to 2000 ng total mass in 200 µL volumes. This equates to sample concentrations of 0.1 – 2000 ng/µL when using 1 – 20 µL sample volumes in a 200 µL total assay volume.

The upper detection limit can be extended to 4000 ng/µL by adding 1 µL of a 4000 ng/µL sample to 199 µL of working reagent. There is some loss of linearity with this assay when adding more than 2000 ng total mass per assay tube.

Kit Contents

Kits are available in 1000, 250 and 50 (evaluation size) assays and include the components in Table 1.

Component1000250EVAL
DeNovix dsDNA Broad Range Dye (100x)2 x 1 mL0.5 mL100 µL
DeNovix dsDNA Broad Range Buffer250 mL50 mL10 mL
DeNovix dsDNA Broad Range Enhancer (100x)2 x 1 mL0.5 mL100 µL
200 ng/µL dsDNA Standard (calf thymus)2 mL1 mL0.5 mL
0 ng/µL dsDNA Standard2 mL1 mL0.5 mL

Best Practices

  • Use calibrated pipettes and DNase-free pipette tips.
  • Prepare the working solution fresh for each assay.
  • Ensure that all samples and standards are treated identically in terms of incubation times and temperature.
  • Avoid introducing air bubbles when mixing.
  • Generate a new standard curve for each assay.
  • Assay total mass must be considered when deciding how much sample to use. This assay is appropriate for 2 – 2000 ng total mass per tube.
  • Label the top, not the sides of the assay tubes.

Sample Prep

  1. Equilibrate all solutions to room temperature before use. Vortex, then centrifuge vials briefly to minimize reagent loss on the cap.
  2. Prepare working solution by mixing 10 mL of the assay buffer with 100 µL of the dye and 100 µL of the enhancer. Scale volumes as needed to make enough volume to aliquot 190 µL of the mixture for each standard and unknown.
  3. For each standard or unknown sample, add 190 µL of the working solution to a labeled tube. Adjust volume when adding more or less than 10 µL of the unknown sample.
  4. Use thin-walled, clear UV-transparent 0.5 mL PCR tubes for assay measurements (DeNovix cat #TUBE-PCR-0.5-500 or equivalent).
  5. Add 10 µL of the 0 ng/µL and 200 ng/µL standards and 1 – 20 µL of unknown DNA samples to the respective tubes and mix well.
  6. Incubate assay tubes at room temperature for 5 minutes.

Sample Measurement

  1. Launch the Fluoro dsDNA app using a DeNovix Fluorometer.
  2. Use the drop-down menu to select the DeNovix dsDNA Broad Range Assay.
  3. Select Preconfigured 2 Standards and then choose Generate New Standard Curve.
  4. Insert the 0 ng/µL dsDNA standard, lower the lid and tap Measure.
  5. Insert the 200 ng/µL dsDNA standard, lower the lid and tap Measure.
  6. After both standards are measured, tap the Samples  button, insert a sample tube and tap Measure.

Reagent Storage

ComponentProtect from LightTemperature
DeNovix dsDNA Broad Range Dye (100x)Yes4°C - Room Temperature
DeNovix dsDNA Broad Range BufferOptional4°C - Room Temperature
DeNovix dsDNA Broad Range Enhancer (100x)Optional4°C - Room Temperature
dsDNA StandardsYes4°C

Revised 19 Oct 2020