产品名称：OligoGreen（效果同OliGreen）ssDNA 定量检测试剂盒 *2000次*

英文名称：OligoGreen ssDNA Assay Kit *2000 assays*

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Introduction

OliGreen® oligonucleotide quantitation reagent is an ultrasensitive fluorescent nucleic acid stain for quantitating oligonucleotides and single-stranded DNA (ssDNA) in solution (Figure 1). Molecular Probes offers the OliGreen reagent either

Figure 1. Linear quantitation of a synthetic 24-mer (an M13 sequencing primer) from 0.1 to 1000 ng/mL using the OliGreen ssDNA quantitation reagent. Samples in 10 mm °? 10 mm cuvettes were excited at 480 nm. The fluorescence emission intensity was measured at 520 nm using a spectrofluorometer and plotted as a function of oligonucleotide concentration. The inset shows an enlargement of the results obtained with oligonucleotide concentrations between zero and 2.0 ng/mL.

as a stand-alone product (O-7582) or as part of a convenient kit (O-11492), which also includes concentrated assay buffer and an oligonucleotide standard. Short, synthetic oligonucleotides are used in a number of molecular biology techniques, such as DNA sequencing, site-directed mutagenesis, DNA amplification and in situ hybridization. Unfortunately, the classic methods for quantitating oligonucleotides are not very sensitive and often require a highly concentrated sample. The most commonly used technique for measuring oligonucleotide and ssDNA concentration is the determination of absorbance at 260 nm (A260). The major disadvantages of the absorbance method are the large relative contribution of nucleotides to the signal, the interference caused by contaminants commonly found in nucleic acid preparations and the relative insensitivity of the assay (an A260 of 0.1 corresponds to ~3 μg/mL solution of a synthetic 24-mer M13 sequencing primer). In contrast, our OliGreen ssDNA quantitation reagent enables researchers to quantitate as little as 100 pg/mL oligonucleotide or ssDNA (200 pg in a 2 mL assay volume) with a standard spectro fluorometer and fluorescein excitation and emission wavelengths. This sensitivity exceeds that achieved with absorbance methods by 10,000-fold. Using a fluorescence microplate reader, we can detect as little as 1 ng/mL oligonucleotide or ssDNA (200 pg in a 200 μL assay volume). We have also quantitated several ssDNAs with the OliGreen reagent, including M13 and fX174 viral DNA and denatured calf thymus DNA, and obtained similar sensitivity. In addition, OliGreen reagent  has been used to detect both phosphodiester andphosphorothioate oligonucleotides in plasma and serum samples1 and to develop a sensitive fluorescence-based capillary electrophoresis method for detecting short, single-stranded oligonucleotides.2 The linear detection range of the OliGreen assay in a standard fluorometer extends over four orders of magnitude in oligonucleotide concentration — from 100 pg/mL to 1 μg/mL — with a single dye concentration (Figure 1). Moreover, we have shown that this linearity is maintained in the presence of several compounds commonly found to contaminate nucleic acid preparations, including salts, urea, ethanol, chloroform, detergents, proteins, ATP and agarose; however, many of these compounds do affect the signal intensity (see Oligonucleotide Standard Curve). Nucleotides and short oligonucleotides of six bases or less do not interfere in the quantitation assay; however, the OliGreen reagent does exhibit fluorescence enhancement when bound to double-stranded DNA (dsDNA) and RNA. Also, we must caution that our experiments with homopolymers have demonstrated that the OliGreen reagent exhibits significant base selectivity. OliGreen dye shows a large fluorescence enhancement when bound to poly(dT), but only a relatively small fluorescence enhancement when bound to poly(dG) and little signal with poly(dA) and poly(dC). Thus, it is important to use an oligonucleotide with similar base composition when generating the standard curve.

Contents and Storage

OliGreen ssDNA Quantitation Reagent

OliGreen ssDNA quantitation reagent is provided as a 1 mL concentrated dye solution in high-quality, anhydrous dimethylsulfoxide (DMSO). Enough reagent is supplied for 200 assays using an assay volume of 2 mL and the protocol described in Experimental Protocol, 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 μL. The DMSO stock solution should be stored frozen at -20°C, desiccated and protected from light. Allow the reagent to warm to room temperature before opening vial. When stored properly, the OliGreen reagent is stable for at least one year.

OliGreen ssDNA Quantitation Kit

OliGreen ssDNA quantitation reagent (Component A),

1 mL solution in DMSO

$ 20X TE (Component B), 25 mL of 200 mM Tris-HCl,

20 mM EDTA, pH 7.5

Oligonucleotide standard (Component C), 1 mL of a 100 μg/mL solution in TE

The OliGreen reagent supplied in the kit is exactly the same as the reagent sold separately; as described above, the DMSO stock solution should be stored frozen at -20°C, desiccated and protected from light. The 20X TE and oligonucleotide standard are best stored at 4°C; however, either may be frozen for longterm storage. The oligonucleotide standard is an 18-base M13 sequencing primer, with the sequence 5￠-TGTAAAACGACGGCCAGT- 3￠. Sufficient material is supplied for 200 assays using a 2 mL assay volume or 2000 assays using a 200 μL assay volume.

Handling and Disposal

Caution: No data are available addressing the mutagenicity or toxicity of OliGreen dsDNA quantitation reagent. Because this reagent binds 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. We strongly recommend using double gloves when handling the DMSO stock solution. As with all nucleic acid reagents, solutions of OliGreen reagent should be poured through activated charcoal before disposal. The charcoal must then be incinerated to destroy the dye.

Experimental Protocol

Overview

The assay procedure is designed for use with standard fluorescence cuvettes and has 2 mL assay volumes. To perform microplate assays, reduce the indicated volumes appropriately. For example, 200 μL volumes are recommended for use with 96-well microplates.

Assay Buffer Preparation

TE assay buffer, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5, is used for diluting the OliGreen reagent and for diluting oligonucleotide and ssDNA samples. Because the OliGreen reagent is an extremely sensitive detection reagent for ssDNA, it is imperative that the TE solution used is free of contaminating nucleic acids. The 20X TE buffer that is included in the OliGreen ssDNA Quantitation Kit is nuclease-free and nucleic acid–free. Prepare the 1X TE working solution, by diluting the concentrated buffer 20-fold with sterile, distilled, DNase-free water.

Reagent Preparation

Immediately before the experiment, prepare an aqueous working solution of the OliGreen reagent by making a 200-fold dilution of the concentrated DMSO solution in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE). For example, to prepare enough working solution to assay 20 samples, add 100 μL OliGreen oligonucleotide quantitation reagent to 19.9 mL TE. We recommend preparing this solution in plastic 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 OliGreen reagent is susceptible to photodegradation. For best results, this solution should be used within a few hours of its preparation.

Oligonucleotide Standard Curve

1.1 Prepare a 2 μg/mL stock solution of oligonucleotide in TE. Determine the oligonucleotide concentration on the basis of absorbance at 260 nm (A260) in a cuvette with a 1 cm pathlength; an A260 of 1.0 corresponds to 30–35 μg/mL oligonucleotide solution. For a standard curve, we commonly use an 18-mer M13 sequencing primer (a 2 μg/mL solution has an A260 of 0.065), although any purified oligonucleotide or ssDNA preparation may be used. The oligonucleotide standard, provided at 100 μg/mL in the OliGreen Kit, can simply be diluted 50-fold in TE to make the 2 μg/mL working solution. It is preferable to prepare the standard curve with oligonucleotides or ssDNA that are similar in length and base composition to the type being assayed. We have found that random-sequence oligonucleotides of ten bases or longer yield approximately equivalent signals, regardless of fragment length; however, OliGreen exhibits significant base selectivity. Our results have also shown that the OliGreen assay remains linear in the presence of several compounds that commonly contaminate nucleic acid preparations, although the signal intensity may be affected (Table 1). Thus, to serve as an effective control, the oligonucleotide or ssDNA 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 five-point standard curve from 10 ng/mL to 1 μg/mL, proceed to step 1.2. For a low-range standard curve from 100 pg/mL to 50 ng/mL, prepare a 20-fold dilution of the 2 μg/mL oligonucleotide solution to yield a 100 ng/mL oligonucleotide stock solution and proceed to step 1.5. 1.2 For the high-range standard curve, dilute the 2 μg/mL oligonucleotide stock solution into disposable 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 OliGreen 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 instrument’s gain should be set so that the sample containing the highest oligonucleotide concentration yields a fluorescence intensity near the fluorometer’s 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 oligonucleotide concentration (see Figure 1). 1.5 For the low-range standard curve — from 100 pg/mL to 50 ng/mL — dilute the 100 ng/mL oligonucleotide stock solution (prepared in step 1.1) 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 OliGreen 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. If necessary, increase the fluorometer gain to accommodate the lower signals by amplifying the fluorescence signal of the low-range standard curve.

Sample Analysis

2.1 Dilute the experimental oligonucleotide solution in TE to a final volume of 1.0 mL in disposable cuvettes or test tubes. You

may wish to prepare more than one dilution of the experimental sample. High dilutions of the experimental sample may serve to

diminish the interfering effect of certain contaminants. However,  extremely small sample volumes should be avoided because they

are difficult to pipet accurately.

2.2 Add 1.0 mL of the aqueous working solution of the OliGreen 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 the standard curve (see steps 1.3 and 1.5). To minimize photobleaching effects, keep the time for fluorescence measurement constant for all samples.

2.4 Subtract the fluorescence value of the reagent blank from that of each of the samples. Determine the oligonucleotide concentra

concentration of the sample from the standard curve generated in Oligonucleotide Standard Curve.

2.5 The assay may be repeated using a different dilution of the

sample to confirm the quantitation results.