Protocol for the quantification of genomic DNA. Provides DNA sample concentration

Qubit dsDNA BR Assay Kit (Qubit dsDNA BR Assay Kit, ThermoFisher, Catalog # Q32850)

• Qubit dsDNA BR Assay Kit Manufacturer’s Instructions
• “The Qubit dsDNA BR Assay Kit is designed specifically for use with the Qubit Fluorometer. The assay is highly selective for double-stranded DNA (dsDNA) over RNA and is designed to be accurate for initial sample concentration from 100 pg/µl–1,000 ng/µl.”

Protocol for Quantification of DNA

• Use only thin-wall, clear, 0.5-mL PCR tubes. Acceptable tubes include Qubit assay tubes (Catalog # Q32856) or Axygen PCR-05-C tubes (Catalog # 10011-830).
• If approximate sample concentration is unknown prior to measurement, run a dilution series with 1-2 samples to bring concentration within range of the assay (e.g., 1:1, 1:10, and 1:100)
• record any dilutions done prior to measurement in order to back calculate original concentration

Procedure

• Label tubes for each of your samples and 2 standards (Label lids only!)
  • Broad Range (standard 1 = 0 ng/μL DNA in TE buffer, standard 2 = 100 ng/μL DNA in TE buffer)
• Prepare the Qubit working solution by diluting the Qubit dsDNA BR Reagent 1:200 in Qubit dsDNA BR Buffer (Volume = #samples + #standards + 1 extra)
  • The final volume in each tube must be 200 μL
  • Each standard tube requires 190 μL of Qubit working solution, and each sample tube requires anywhere from 180–199 μL
  • Prepare sufficient Qubit working solution to accommodate all standards and samples
• Add 190μL of working solution to each of the tubes used for standards
• Add 10μL of each standard to the appropriate tube, then mix by vortexing for 2–3 seconds (Take care NOT to create bubbles)
• Add Xµl of working solution to individual assay tubes so that the final volume in each tube after adding sample is 200 μL (no less than 180µl)
• Add 200µl - Xµl of sample to each tube, then mix by vortexing for 2–3 seconds (Take care NOT to create bubbles)
• Incubate tubes at room temperature for 2min (cover with foil)
• Read standards on Qubit
• Set volume of sample and readout in ng/µl and read each tube