Checking DNA concentration with the Qubit fluorometer

The Qubit fluorometer allows for precise measurement of the concentration of double-stranded DNA in a sample. This is done by intercalating dye in between the bases in the strands. For this reason, it is widely accepted that the Qubit is more accurate than a spectrophotometer, which cannot distinguish between RNA, ssDNA, and dsDNA.

Qubit Fluorometer Diagram

  1. Gather the following materials:
    • CCG 1X dsDNA High Sensitivity Assay Kit (working solution + two standards)
    • (2) Qubit assay tubes (for standards), labeled for standards 1 and 2
    • (1) Qubit assay tube per sample to be assayed, labeled
  2. Sign out the number of tubes used (including standards) on the sheet next to the Qubit station.
  3. Prepare working solution using filter tips into Qubit assay tubes (in drawer) as follows:
    • Standards: 190 µL of working solution + 10 µL standard = 200 µL
    • Samples: 199* µL of working solution + 1 µL* of sample = 200 µL

199 µL of working solution and 1 µL of sample is the standard amount to use unless samples are suspected to be at very low or very high concentrations. For suspected low concentrations, input can be a range of 180-199 µL of working solution + 1-20 µL sample, for a final volume of 200 µL. For suspected high concentrations (>60 ng/µL), dilute your DNA (1:10 or 1:100).

  1. Vortex assay tubes for 3 seconds. Let sit for 2 min.
  2. Turn on the Qubit and select the appropriate assay and kit. Press ‘yes’ to read new standards and place the mix of standard 1 into the tube receptacle and press ‘read standard’. Repeat for standard 2.
  3. Continue by reading your samples one by one. Be mindful of the units being reported and change if desired. If using a Qubit 1 or 2, be sure to use the Dilution Calculator feature to determine stock concentration of your original sample.

Dilution Calculator instructions - after reading the sample, press ‘calculate stock volume’, change the amount to ‘1 µL’ or how much DNA you added to your sample tube, and record that value. Press ‘read next sample’ for the next tube. Repeat as needed.

  1. We recommend, concentrations be manually recorded. Reports can be exported from the Qubit using a flash drive, but sample names are not recorded, so you must be careful to remember the order you read samples or record that separately.

Please ask Athena or Grace if you need to order a different Qubit kit, such as Broad Range DNA or RNA kits.