Quant-iT PicoGreen dsDNA Reagent and Kits

Table 1. Contents and Storage Information.

Material Quant-iT PicoGreen dsDNA reagent (Component A) 20X TE (Component B) Lambda DNA standard (Component C)

Amount 1 mL in 1 vial (P7589) or in10 vials of 100 L each (P11496) 25 mL 1 mL

Concentration Solution in DMSO 200 mM Tris-HCl, 20 mM EDTA, pH 7.5 100 g/mL in TE

Storage 26C Desiccate Protect from light Room temperature * 26C *

Stability

When stored as directed, product stable for at least 6 months

* For long-term storage, both the 20X TE and lambda DNA standard can be stored at 20C. Number of Labelings: For either the kits or the stand-alone reagent, sufficient reagent is supplied for 200 assays using an assay volume of 2 mL and the protocol described below. Note that the assay volume is dependent on the instrument used to measure fluorescence; with a microplate reader and a 96-well microplate, the assay volume is reduced to 200 L and 2,000 assays are possible. Spectral Data: Quant-iT PicoGreen dsDNA reagent 502/523 nm, bound to nucleic acids

Introduction
Quant-iT PicoGreen dsDNA 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 diagnostic techniques, such as quantitating DNA amplification products and detecting DNA molecules in drug preparations. The Quant-iT PicoGreen reagent has recently been used to quantitate PCR amplification yields in a method for direct cycle sequencing of PCR products.1 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 and single-stranded nucleic acids 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 5 g/mL dsDNA solution). Hoechst (bisbenzimide) dyes are sensitive fluorescent nucleic acid stains that circumvent many of these problems. The Hoechst 33258based 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.2 Molecular Probes proprietary YO-PRO-1 and YOYO-1 nucleic acid stains have also been used to quantitate nucleic acids, allowing the detection of about 0.5 ng/mL DNA in solution.3,4

Revised: 10June2008

MP 07581

Our Quant-iT PicoGreen dsDNA reagent enables researchers to quantitate as little as 25 pg/mL of dsDNA (50 pg dsDNA in a 2 mL assay volume) with a standard spectrofluorometer and fluorescein excitation and emission wavelengths. This sensitivity exceeds that achieved with the Hoechst 33258based assay by 400-fold. Using a fluorescence microplate reader, we can detect as little as 250 pg/mL dsDNA (50 pg in a 200 L assay volume). The standard Quant-iT PicoGreen assay protocol is also simpler than that for Hoechst 33258 because a single concentration of the Quant-iT 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 Quant-iT PicoGreen assay in a standard fluorometer extends over more than four orders of magnitude in DNA concentrationfrom 25 pg/mL to 1,000 ng/mLwith a single dye concentration (Figure 1). We have shown that this linearity is maintained in the presence of several compounds that commonly contaminate nucleic acid preparations, including salts, urea, ethanol, chloroform, detergents, proteins, and agarose. Our assay protocol was also developed to minimize the fluorescence contribution of RNA and single-stranded DNA (ssDNA) (Figure 2). Although the Hoechst 33258based method is not significantly affected by the presence of RNA when the assay is carried out in the recommended high-salt buffer, we have found that Hoechst 33258 does exhibit a large fluorescence enhancement with ssDNA under these conditions. Furthermore, when the Hoechst 33258 based assay is carried out in TE alone (10 mM Tris-HCl, 1 mM EDTA, pH 7.5, with no NaCl added), RNA contributes a significant fluorescence signal. Using the Quant-iT PicoGreen dsDNA reagent and the recommended assay protocol, researchers can quantitate dsDNA in the presence of equimolar concentrations of ssDNA and RNA with minimal effect on the quantitation results.

Figure 1. Dynamic range and sensitivity of the Quant-iT PicoGreen dsDNA assay. Calf thymus DNA was added to cuvettes containing Quant-iT PicoGreen dsDNA reagent diluted in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE). The samples were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm using a spectrofluorometer. Fluorescence emission intensity was then plotted versus DNA concentration; the inset shows an enlargement of the results obtained with DNA concentrations between zero and 750 pg/mL.

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Figure 2. Fluorescence enhancement of Quant-iT PicoGreen reagent upon binding dsDNA, ssDNA, and RNA. Samples containing 500 ng/mL calf thymus DNA, M13 ssDNA, or E. coli ribosomal RNA were added to cuvettes containing Quant-iT PicoGreen reagent in TE. Samples were excited at 480 nm and the fluorescence emission spectra were collected using a spectrofluorometer. Emission spectra for samples containing dye and nucleic acids, as well as for dye alone (baseline), are shown.

Before You Begin

The Quant-iT PicoGreen reagent supplied in the kits is exactly the same as the reagent sold separately.

Handling and Disposal

Allow the Quant-iT PicoGreen reagent to warm to room temperature before opening the vial.

Caution:

No data are available addressing the mutagenicity or toxicity of Quant-iT PicoGreen dsDNA reagent. Because this reagent binds to nucleic acids, it should be treated as a potential mutagen and handled with appropriate care. The DMSO stock solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues. Dispose of Quant-iT PicoGreen reagent in accordance with local regulations.

Preparing the Assay Buffer

TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is used below for diluting the Quant-iT PicoGreen reagent, for diluting DNA samples, and in the assay itself. Because the Quant-iT PicoGreen dye is an extremely sensitive detection reagent for dsDNA, it is imperative that the TE solution used be free of contaminating nucleic acids. The 20X TE buffer included in the Quant-iT PicoGreen dsDNA Assay Kits is certified to be nucleic acidfree and DNasefree. Prepare a 1X TE working solution by diluting the concentrated buffer 20-fold with sterile, distilled, DNase-free water.

Preparing the Reagent

On the day of the experiment, prepare an aqueous working solution of the Quant-iT PicoGreen reagent by making a 200-fold dilution of the concentrated DMSO solution in TE. For example, to prepare enough working solution to assay 20 samples in a 2 mL final volume, add 100 L Quant-iT PicoGreen dsDNA reagent to 19.9 mL TE. We recommend preparing this solution in a plastic container rather than glass, 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 Quant-iT PicoGreen reagent is susceptible to photodegradation. For best results, this solution should be used within a few hours of its preparation.

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Experimental Protocol
DNA Standard Curve

1.1 Prepare a 2 g/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 2 g/mL dsDNA solution. For a standard curve, we commonly use bacteriophage lambda or calf thymus DNA, although any purified dsDNA preparation may be used. The lambda DNA standard, provided at 100 g/mL in the Quant-iT PicoGreen Kits, can simply be diluted 50-fold in TE to make the 2 g/mL working solution. For example, 30 L of the DNA standard mixed with 1.47 mL of TE will be sufficient for the standard curve described below. It is sometimes preferable to prepare the standard curve with DNA similar to the type being assayed; e.g., long or short linear DNA fragments when quantitating similarsized restriction fragments or plasmid when quantitating plasmid DNA. However, we have found that most linear dsDNA molecules yield approximately equivalent signals, regardless of fragment length. Our results have shown that the Quant-iT PicoGreen assay remains linear in the presence of several compounds that commonly contaminate nucleic acid preparations, although the signal intensity may be affected (Table 2). 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 create a five-point standard curve from 1 ng/mL to 1 g/mL, proceed to step 1.2. For a low-range standard curve from 25 pg/mL to 25 ng/mL, prepare a 40-fold dilution of the 2 g/mL DNA solution to yield a 50 ng/mL DNA stock solution and proceed to step 1.5. 1.2 For the high-range standard curve, dilute the 2 g/mL DNA stock solution into disposable cuvettes (or plastic test tubes for transfer to quartz cuvettes) as shown in Table 3. Then add 1.0 mL of the aqueous working solution of Quant-iT PicoGreen reagent (prepared in Reagent Preparation) to each cuvette. Mix well and incubate for 2 to 5 minutes at room temperature, protected from light. 1.3 After incubation, measure the sample fluorescence using a spectrofluorometer or fluorescence microplate reader and standard fluorescein wavelengths (excitation ~480 nm, emission ~520 nm). To ensure that the sample readings remain in the detection range of the fluorometer, the instruments gain should be set so that the sample containing the highest DNA concentration yields a fluorescence intensity near the fluorometers maximum. To minimize photobleaching effects, keep the time for fluorescence measurement constant for all samples. 1.4 Subtract the fluorescence value of the reagent blank from that of each of the samples. Use corrected data to generate a standard curve of fluorescence versus DNA concentration (see Figure 1). 1.5 For the low-range standard curvefrom 25 pg/mL to 25 ng/mLdilute the 50 ng/mL DNA stock solution (prepared in step 1.1) into disposable cuvettes (or plastic test tubes for transfer to quartz cuvettes) as shown in Table 4. Add 1.0 mL of the aqueous working solution of Quant-iT PicoGreen reagent (prepared in Reagent Preparation) to each cuvette. Mix well and incubate for 2 to 5 minutes at room temperature, protected from light. Continue with steps 1.3 and 1.4. Adjust the fluorometer gain to accommodate the lower fluorescence signals.

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Table 2. Effects of several compounds that commonly contaminate nucleic acid preparations on the signal intensity of the Quant-iT PicoGreen dsDNA assay.

Compound
Salts Ammonium acetate Sodium acetate Sodium chloride Zinc chloride Magnesium chloride Urea Organic Solvents Phenol Ethanol Chloroform Detergents Sodium dodecyl sulfate Triton X-100 Proteins Bovine serum albumin IgG Other Compounds Polyethylene glycol Agarose

Maximum Acceptable Concentration

50 mM 30 mM 200 mM 5 mM 50 mM 2M 0.1% 10% 2% 0.01% 0.1% 2% 0.1% 2% 0.1%

% Signal Change*

3% decrease 3% increase 30% decrease 8% decrease 33% decrease 9% increase 13% increase 12% increase 14% increase 1% decrease 7% increase 16% decrease 19% increase 8% increase 4% increase

* The compounds were incubated at the indicated concentrations with Quant-iT PicoGreen reagent in the presence of 500 ng/mL calf thymus DNA. All samples were assayed in a final volume of 200 L in 96-well microplates using a CytoFluor microplate reader. Samples were excited at 485 nm and fluorescence intensity was measured at 520 nm.

Table 3. Protocol for preparing a high-range standard curve.

Volume (L) of TE
0 900 990 999 1,000

Volume (L) of 2 g/mL DNA Stock

1,000 100 10 1 0

Volume (L) of Diluted Quant-iT PicoGreen Reagent

1,000 1,000 1,000 1,000 1,000

Final DNA Concentration in Quant-iT PicoGreen Assay

1 g/mL 100 ng/mL 10 ng/mL 1 ng/mL blank

Sample Analysis

2.1 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 may diminish the interfering effect of certain contaminants. However, extremely small sample volumes should be avoided because they are difficult to pipet accurately. See Eliminating Single-Stranded Nucleic Acids from Samples (below) for information on eliminating RNA and ssDNA from the sample. 2.2 Add 1.0 mL of the aqueous working solution of the Quant-iT PicoGreen reagent to each sample. Incubate for 2 to 5 minutes at room temperature, protected from light. 2.3 Measure the fluorescence of the sample using instrument parameters that correspond to those used when generating your standard curve (see step 1.3). To minimize photobleaching effects, keep the time for fluorescence measurement constant for all samples.

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2.4 Subtract the fluorescence value of the reagent blank from that of each of the samples. Determine the DNA concentration of the sample from the standard curve generated in DNA Standard Curve. 2.5 The assay may be repeated using a different dilution of the sample to confirm the quantitation results.

Eliminating Single-Stranded Nucleic Acids from Samples

We have found that dsDNA can be quantitated in the presence of equimolar concentrations of single-stranded nucleic acids with minimal interference. Table 5 shows the concentrations of RNA or ssDNA that, for a given dsDNA concentration, result in less than a 10% change in the signal intensity using the Quant-iT PicoGreen assay protocol. Fluorescence due to Quant-iT PicoGreen reagent binding to RNA at high concentrations can be eliminated by treating the sample with DNase-free RNase.5 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.5

Table 4. Protocol for preparing a low-range standard curve.

Volume (L) of TE
0 900 990 999 1,000

Volume (L) of 50 ng/mL DNA Stock

1,000 100 10 1 0

Volume (L) of Diluted Quant-iT PicoGreen Reagent

1,000 1,000 1,000 1,000 1,000

Final DNA Concentration in Quant-iT PicoGreen Assay

25 ng/mL 2.5 ng/mL 250 pg/mL 25 pg/mL blank

Table 5. Sensitivity of the Quant-iT PicoGreen dsDNA assay for quantitating dsDNA in the presence of singlestranded nucleic acids.

[dsDNA]*
1 g/mL 500 ng/mL 10 ng/mL 5 ng/mL 100 pg/mL 50 pg/mL

[RNA] (amount relative to dsDNA)

10 g/mL 500 ng/mL 100 ng/mL 50 ng/mL 1 ng/mL 500 pg/mL (10X) (1X) (10X) (10X) (10X) (10X)

[ssDNA] (amount relative to dsDNA)

300 ng/mL 50 ng/mL 30 ng/mL 15 ng/L 1 ng/mL 500 pg/mL (0.3X) (0.1X) (3X) (3X) (10X) (10X)

* For several concentrations of dsDNA, we show the concentration of RNA or ssDNA that results in no more than a 10% increase in the samples signal intensity.

References
1. Biotechniques 20, 676 (1996); 2. Anal Biochem 102, 344 (1980); 3. Anal Biochem 208, 144 (1993); 4. Biophys J 61, A314 (1992); 5. Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).

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Product List
Cat # P7589 P11496 P7581 P11495

Current prices may be obtained from our website or from our Customer Service Department.

Product Name Unit Size Quant-iT PicoGreen dsDNA Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kit Quant-iT PicoGreen dsDNA Assay Kit *10 100 L*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kit Quant-iT PicoGreen dsDNA reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL Quant-iT PicoGreen dsDNA reagent *10 100 L*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 100 L

Contact Information
Molecular Probes, Inc. 29851 Willow Creek Road Eugene, OR 97402 Phone: (541) 465-8300 Fax: (541) 335-0504 Customer Service: 6:00 am to 4:30 pm (Pacific Time) Phone: (541) 335-0338 Fax: (541) 335-0305 probesorder@invitrogen.com Toll-Free Ordering for USA: Order Phone: (800) 438-2209 Order Fax: (800) 438-0228 Technical Service: 8:00 am to 4:00 pm (Pacific Time) Phone: (541) 335-0353 Toll-Free (800) 438-2209 Fax: (541) 335-0238 probestech@invitrogen.com Invitrogen European Headquarters Invitrogen, Ltd. 3 Fountain Drive Inchinnan Business Park Paisley PA4 9RF, UK Phone: +44 (0) 141 814 6100 Fax: +44 (0) 141 814 6260 Email: euroinfo@invitrogen.com Technical Services: eurotech@invitrogen.com Further information on Molecular Probes products, including product bibliographies, is available from your local distributor or directly from Molecular Probes. Customers in Europe, Africa and the Middle East should contact our office in Paisley, United Kingdom. All others should contact our Technical Service Department in Eugene, Oregon. Molecular Probes products are high-quality reagents and materials intended for research purposes only. These products must be used by, or directly under the supervision of, a technically qualified individual experienced in handling potentially hazardous chemicals. Please read the Material Safety Data Sheet provided for each product; other regulatory considerations may apply. Limited Use Label License No. 223: Labeling and Detection Technology For research use only. Not intended for any animal or human therapeutic or diagnostic use. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) to not transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, 29851 Willow Creek Road, Eugene, OR 97402. Tel: (541) 465-8300. Fax: (541) 335-0504. Several Molecular Probes products and product applications are covered by U.S. and foreign patents and patents pending. All names containing the designation are registered with the U.S. Patent and Trademark Office. Copyright 2008, Molecular Probes, Inc. All rights reserved. This information is subject to change without notice.

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