TD-700 Fluorometer Method for dsDNA Quantitation using
PicoGreen®

Introduction

PicoGreen dsDNA Quantitation Reagent is an ultrasensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in solution. Detecting and quantitating small amounts of DNA is extremely important in a wide variety of biological applications. These include standard molecular biology techniques, such as synthesizing cDNA for library production and purifying DNA fragments for subcloning, as well as techniques, such as quantitating DNA amplification products and detecting DNA molecules in drug preparations.

The most commonly used technique for measuring nucleic acid concentration is the determination of absorbance at 260 nm (A260) The major disadvantages of the absorbance method are the large relative contribution of nucleotides, single-stranded nucleic acids and proteins to the signal, the interference caused by contaminants commonly found in nucleic acid preparations, the inability to distinguish between DNA and RNA, and the relative insensitivity of the assay (an A260 of 0.1 corresponds to a 5µg/mL dsDNA solution). Hoechst (bisbenzimide) dyes are sensitive fluorescent nucleic acid stains that circumvent many of these problems. The Hoechst 33258 Dye - based assay is somewhat selective for dsDNA, does not show significant fluorescence enhancement in the presence of proteins and allows the detection and quantitation of DNA concentrations as low as 10 ng/mL DNA.(1)

The Turner BioSystems TD-700 Laboratory Fluorometer used in conjunction with Molecular Probes' PicoGreen dsDNA Quantitation Reagent enables researchers to quantitate as little as 25 pg/mL of dsDNA (50 pg dsDNA in a 2-mL assay volume). This sensitivity exceeds that achieved with the Hoechst 33258 Dye - based assay by 400-fold.

The standard PicoGreen assay protocol is also simpler than the Hoechst 33258 Dye method, because a single concentration of the PicoGreen Reagent allows detection over the full dynamic range of the assay. In order to achieve more than two orders of magnitude in dynamic range with Hoechst-based assays, two different dye concentrations are recommended. In contrast, the linear detection range of the PicoGreen assay in the TD-700 Fluorometer extends over more than four orders of magnitude in DNA concentration - from 25 pg/mL to 1000 ng/mL - with a single dye concentration (see figures). This linearity is maintained in the presence of several compounds commonly found to contaminate nucleic acid preparations, including salts, urea, ethanol, chloroform, detergents, proteins and agarose. The assay protocol has been developed to minimize the fluorescence contribution of RNA and single-stranded DNA (ssDNA). Using the PicoGreen dsDNA Quantitation Reagent and the TD-700 Fluorometer, researchers can quantitate dsDNA in the presence of equimolar concentrations of ssDNA and RNA with minimal effect on the quantitation results.

2. Materials Required

• TD-700 Fluorometer with standard PMT and 10 x 10 mm square cuvette adaptor (P/N 7000-009)
• Fluorescein filter kit (P/N 10-086R) which includes 486 nm excitation filter (P/N 10-105) and 510-700 emission filter (P/N 10-109R-C) and two Blue Mercury Vapor lamps (P/N 10-089).
• 10 x 10 mm square polystyrene disposable cuvettes (P/N 7000-957)
• PicoGreen dsDNA Quantitation Reagent, supplied by Molecular Probes, Inc., Eugene, Oregon, catalog number P-7581. A single 1-mL unit of the reagent concentrate is sufficient for 200 assays using an assay volume of 2 mL and the protocol described in section 3. Handling, storage and use of the reagent should be performed in accordance with the product information sheet supplied by Molecular Probes, Inc.

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3. Experiment Protocol

3.1 Reagent Preparation

The PicoGreen dsDNA Quantitation Reagent is supplied as a 1-mL concentrated dye solution in anhydrous dimethylsulfoxide (DMSO). On the day of the experiment, prepare an aqueous working solution of the PicoGreen Reagent by making a 1:200 dilution of the concentrated dye solution in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE). To prepare enough working solution to assay 20 samples, add 100 µL PicoGreen dsDNA Quantitation Reagent to 20.0 mL TE. Preparing this solution in a plastic container is recommended, as the reagent may adsorb to glass surfaces. Protect the working solution from light by covering it with foil or placing it in the dark, as the PicoGreen Reagent is susceptible to photodegradation. For best results, this solution should be used within a few hours of its preparation.

3.2 DNA Standard Curve

3.2.1 Prepare a 2 µg/mL stock solution of dsDNA in TE. Determine the DNA concentration on the basis of absorbance at 260 nm (A260) in a cuvette with a 1-cm pathlength; an A260 of 0.04 corresponds to 2µg/mL dsDNA solution. Calf thymus DNA is commonly used for a standard curve, although any purified dsDNA preparation may be used. It is preferable to prepare the standard curve with DNA similar to the type being assayed; long or short linear DNA fragments for quantitating similar-sized restriction fragments; plasmid for quantitating plasmid DNA. However, most linear dsDNA molecules have been found to yield approximately equivalent signals, regardless of fragment length. The PicoGreen assay remains linear in the presence of several compounds that commonly contaminate nucleic acid preparations, although the signal intensity may be affected. Thus, to serve as an effective control, the dsDNA solution used to prepare the standard curve should be treated the same way as the experimental samples and should contain similar levels of such compounds.

To generate a single-replicate, five-point standard curve from 1 ng/mL to 1 µg/mL, proceed to step 3.2.2. For a low-range standard curve from 25 pg/mL to 25 ng/mL, prepare a 40-fold dilution of the 2 µg/mL DNA solution to yield a 50 ng/mL DNA stock solution and proceed to step 3.2.5.

3.2.2 For the high-range standard curve, dilute the 2µg/mL DNA stock solution into disposable cuvettes (or alternatively, into plastic test tubes for transfer to quartz cuvettes) as shown in Table 1. Then add 1.0 mL of the aqueous working solution of PicoGreen Reagent (prepared in section 3.1) to each cuvette. Mix well and incubate for 2 to 5 minutes at room temperature, protected from light.

Table 1. Protocol for preparing high-range standard curve.

3.2.3 After incubation, measure the sample fluorescence in the TD-700 Fluorometer using the fluorescein filter kit (P/N 10-086R). Insert the most fluorescent sample first (1 µg/mL DNA) and calibrate the instrument sensitivity as directed in the TD-700 Operating Manual (press #2, calibrate). This procedure automatically optimizes the instrument sensitivity to match the fluorescence of the sample.

Figure 1A . High-range calibration plot.

Figure 1B: Low range calibration plot.

Figure 1C: Enlargement of lower left segment of Figure 1B.

Figures: High-range (1A) and low-range (1B) calibration plots for quantitation of calf thymus DNA on the TD-700 fluorometer. Figure 1C shows an enlargement of the area indicated by the box in the lower left hand corner of Figure 1B.

3.2.4 Measure the fluorescence of the remaining samples. To equalize any photobleaching effects, insert samples into the fluorometer for approximately equal time periods. The fluorescence value of the reagent blank may be subtracted from that of each sample. Corrected or uncorrected data may be used to generate a standard curve of fluorescence versus DNA concentration.

3.2.5 For the low-range standard curve - from 25 pg/mL to 25 ng/mL - dilute the 50 ng/mL DNA stock solution (prepared in step 3.2.1) into dispoable cuvettes (or plastic test tubes for transfer to quartz cuvettes) as shown in Table 2. Then add 1.0 mL of the aqueous working solution of PicoGreen Reagent (prepared in section 3.1) to each cuvette. Mix well and incubate for 2 to 5 minutes at room temperature, protected from light. Insert the most fluorescent (25 ng/mL) sample first and adjust the fluorometer sensitivity factor (as in 3.2.3), to accommodate the lower fluorescence signals. Measure the fluorescence of the remaining samples. Plot a low-range standard curve (Figure 1B,C) after optionally subtracting the reagent blank fluorescence value. A

A. The fluorescence signal at the detection limit of 25 pg/mL dsDNA is typically about 3% above background; however the high precision of the assay data makes this low detection limit readily achievable in practice.

3.3 Sample Analysis

3.3.1 Dilute the experimental DNA solution in TE to a final volume of 1.0 mL in disposable cuvettes or test tubes. You may alter the amount of sample diluted, provided that the final volume remains 1.0 mL. A higher dilution of the experimental sample will ensure that any contaminants are maximally diluted. However, extremely small sample volumes should be avoided because they are difficult to pipet accurately. See section 3.4 for information on eliminating RNA and ssDNA from the sample.

Table 2. Protocol for preparing low-range standard curve.

3.3.2 Add 1.0 mL of the aqueous working solution of the PicoGreen Reagent (prepared in section 3.1) to each sample. Incubate for 2 to 5 minutes at room temperature, protected from light.

3.3.3 Measure the fluorescence of the sample using instrument parameters that correspond to those used when generating your standard curve (see steps 3.2.3, 3.2.4 and 3.2.5). To equalize any photobleaching effects, insert samples into the fluorometer for approximately equal time periods.

3.3.4 If the standard curve was plotted using blank-subtracted data (section 3.2.4), the reagent blank fluorescence value must also be subtracted from that of each of the samples. Determine the DNA concentration of the sample from the standard curves generated in section 3.2.

3.3.5 The assay may be repeated using a different dilution of the sample to confirm the quantitation results.

3.4 Eliminating Single-Stranded Nucleic Acids from Samples

Double-stranded DNA can be quantitated in the presence of equimolar concentrations of single-stranded nucleic acids with minimal interference. A 10-fold excess of RNA over dsDNA generally produces no more than a 10% change in the fluorescence signal. Somewhat larger distortions are produced by ssDNA, particularly at low DNA concentrations (see Molecular Probes' product information sheet MP7581 for more details). Fluorescence due to PicoGreen Reagent binding to RNA at high concentrations can be eliminated by treating the sample with DNase-free RNase. (2)The use of RNase A/RNase T1 with S1 nuclease will eliminate all single-stranded nucleic acids and ensure that the entire sample fluorescence is due to dsDNA.(2)

4. References

1. Anal. Biochem. 102, 344 © 1980
2. Molecular Cloning: A Laboratory Manual, Second Edition, J. Sambrook, E.F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York © 1989.

The PicoGreen dsDNA Quantitation Reagent is the subject of patent applications filed by Molecular Probes, Inc. and is not available for resale or other commercial uses without a specific agreement from Molecular Probes, Inc. PicoGreen is a registered trademark of Molecular Probes, Inc.

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