Quant-iT™ Protein Assay Kit

Negative fluorescence is a physical impossibility. It is an artifact from software autocorrecting for background signal. This means your reader is picking up and subtracting out background light at the cost of your data. Make sure to do a buffer-only control and assess the type of signal. You may need to switch to a different plate.

Yes, the manual has directions for this application. You will use the 0 ng/µL lambda dsDNA HS standard to generate Standard #1. You will prepare a dilution of the 10 ng/µL lambda dsDNA HS standard to generate Standard #2. You then prepare the samples and compare them to this 2-point standard curve. The Quant-iT dsDNA BR Kit can be used in a similar manner.

PicoGreen dye and other fluorescence-based quantification reagents are not recommended for quantifying dye-conjugated nucleic acids. The attached dye molecules can interfere with either binding and/or fluorescence output of the quantification reagents.

Strands that are roughly in the 20-mer range or shorter show a lower level of signal. For dsDNA samples that are composed of mostly short strands, the reagent may still be used, but one should use a dsDNA standard that is of comparable length as the sample.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.

The Qubit Fluorometer contains highly optimized algorithms that calculate the concentration of the sample using either the Qubit assays or the Quant-iT DNA assays. The Quant-iT PicoGreen DNA assay may be adapted to the Qubit Fluorometer using the MyQubit firmware. The performance of all of these assays is similar.

The Quant-iT PicoGreen DNA assay is the most established assay and the most general-purpose (http://tools.thermofisher.com/content/sfs/manuals/PicoGreen-dsDNA-protocol.pdf). It requires the dilution of the standard DNA and buffer but can be adapted for use with either cuvettes, microplates, or the NanoDrop 3300.

The Quant-iT DNA assays provide a ready-to-use buffer and pre-diluted standard DNA for analyzing a large number of samples (>20 samples) using a 96-well microplate with no further adaptation.

The Qubit assays (https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-assays/myqubit.html) are intended for low throughput (<20 samples), and are only used on the Qubit Fluorometer.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.