Quant-iT™ PicoGreen™ dsDNA Reagent, 1 mL - FAQs

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.

The buffer included in the kit should assure the proper pH range, even if your DNA is at a pH outside of this range, since at least a 10-fold excess of kit buffer over sample is used in the assay.

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.

Yes, we would recommend reducing the volume while keeping the dye: sample ratio constant.

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.

Yes, you can, for Qubit instruments developed after the original Qubit (1.0) Fluorometer. See MyQubit assay instructions here (http://www.thermofisher.com/us/en/home/life-science/laboratory-instruments/fluorometers/qubit/qubit-assays/myqubit.html.html).

Generally, the cleaner the sample the better. Some salts, proteins, and detergents are tolerated in the assays; see the specific assay protocol for which ones and at what concentrations.

Several nucleic acid stains will label mitochondrial DNA as well as nuclear DNA. One stands out above the rest: PicoGreen stain. It will label nuclear DNA as well, but the mitochondrial DNA label stands out in cells that are adherent and relatively large. See the publication: Exp Cell Res. (2005) 303(2):432-46 .

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

The accuracy and sensitivity of the Qubit quantitation assays are the same as that of a microplate reader. This was a requirement during product development. The detection limits for each Qubit kit can be found on the corresponding product manual, which can be found by searching our website by keyword or catalog number.

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

No. The Qubit DNA and RNA kits only quantify the amount of either DNA or RNA in the sample. The Qubit fluorometer cannot take absorbance readings to provide a A260/A280 ratio or detect protein in nucleic acid samples. This can be done with the NanoDrop instrument. If your sample contains protein or other contaminants that can affect the assay, it should be further purified.

If your sample may contain both DNA and RNA, one may use either (or both) the DNA and RNA Qubit kits and compare with samples treated with either RNase or DNase to get an accurate determination of DNA or RNA, respectively.

All Quant-iT and Qubit kits are compatible with all fluorometers and microplate readers that have the appropriate light sources and filters. You won't have access to the algorithm in the Qubit fluorometer for generating the standard curve provided by the instrument, instead, you must make a few dilutions of the highest standard DNA or RNA (Standard #2) in the Qubit kits to generate a standard curve with multiple data points.

No, we do not recommend this. Some of the dyes in the original Quant-iT kits (those NOT listed as “for use with the Qubit fluorometer”) are not compatible with the Qubit Fluorometer. In addition, the new Quant-iT kits (labeled as “for use with the Qubit Fluorometer”) have standards formulated to be compatible with the Qubit fluorometer internal algorithms for the respective assays. The Qubit Fluorometer-compatible kits are also less expensive per assay if you are processing fewer than 20 samples at a time.