Technical Note 144

High Sensitivity Assay Standard Protocol

Introduction

The DeNovix® dsDNA High Sensitivity Assay enables the accurate detection of purified double-stranded DNA (dsDNA) samples with a standard detection range from 100 pg to 250 ng total mass in 200 µL volumes. This equates to sample concentrations of 10 pg/µL – 250 ng/µL when using between 1 – 20 µL sample volumes in 200 µL assay volumes. The assay is linear for sample concentrations as high as 250 ng/µL when adjusting volumes to 1 µL of sample into 199 µL of working reagent. Total mass should not exceed 250 ng for best results.

The lower detection limit can be extended down to 5 pg/µL sample by adding 20 µL of the 5 pg/µL sample to 180 µL of the working reagent.

Kit Contents

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

Component1000250EVAL
DeNovix dsDNA High Sensitivity Dye (100x)2 x 1 mL0.5 mL100 µL
DeNovix dsDNA High Sensitivity Buffer200 mL50 mL10 mL
25 ng/µL dsDNA Standard (calf thymus)2 mL1 mL0.5 mL
0 ng/µL dsDNA Standard2 mL1 mL0.5 mL

Best Practices

  • Use properly calibrated pipettes and DNase-free pipette tips.
  • Treat all standards and samples identically in terms of incubation times and temperature.
  • Avoid introducing air bubbles when mixing.
  • Generate a new standard curve for each assay.
  • Ensure that sample solution contaminant levels are compatible with the assay.
  • Assay total mass must be considered when deciding how much sample to use. This assay is appropriate for 100 pg – 250 ng total mass per tube.
  • Label only the tops of the tubes.

Sample Prep

  1. Allow all solutions to equilibrate to room temperature before use. Vortex, then centrifuge vials briefly before opening 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. Scale volumes as needed to make enough volume to aliquot 190 µL of the mixture for each standard and unknown. Discard after 24 hours.
  3. For each standard or unknown sample, add 190 µL of the working solution into a labeled tube. Adjust volume when adding more or less than 10 µL of the unknown sample.
  4. Use thin-walled, clear 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, 25 ng/µL standards or 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 High Sensitivity Assay.
  3. Select Preconfigured 2 Standards and then choose Generate New Standard Curve.
  4. Insert the 0 ng/µL dsDNA standard tube, lower the lid and tap the Measure button.
  5. Insert the 25 ng/µL dsDNA standard tube, lower the lid and tap the Measure button.
  6. After both standards are measured, tap the Samples button, insert a sample tube and tap Measure.

Reagent Storage

ComponentProtect from LightTemperature
DeNovix dsDNA High Sensitivity Dye (100x)Yes4oC - Room Temperature
DeNovix dsDNA High Sensitivity BufferOptional4oC - Room Temperature
dsDNA StandardsYes4oC

Revised 19 Oct 2020