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A Modulus™ Microplate Fluorometer Method for Quantitation of ssDNA Using
1. INTRODUCTION Quant-iT™ OliGreen® oligonucleotide reagent is an ultra-sensitive fluorescent nucleic acid stain. Use of OliGreen allows performance of simple and rapid procedures for quantitating oligonucleotides and single-stranded DNA (ssDNA). Short, synthetic oligonucleotides are used in a number of molecular biology techniques, including: DNA sequencing, site-directed mutagenesis, DNA amplification, and in situ hybridization. The most commonly used technique for measuring oligonucleotide and ssDNA concentration is by determining absorbance at 260 nm. Major disadvantages of this method are poor sensitivity and interference in signal levels from contaminating components such as nucleotides, proteins, and salts in the RNA solution. The use of an ultra-sensitive fluorescent nucleic acid stain such as OliGreen® alleviates these problems. The Turner BioSystems Modulus™ Microplate Fluorometer used in conjunction with the Molecular Probes OliGreen® ssDNA Reagent Kit allows for rapid and accurate measurement of ssDNA concentrations in small-volume microplates (200 µL per well). As little as 100 pg of ssDNA can be quantitated using the Blue Fluorescent Optical Kit configuration of the Modulus™ Microplate Fluorometer. The linear dynamic range extends over 4 orders of magnitude from 100 pg/mL - 1 µg/mL ssDNA (Figure 1). 2. MATERIALS REQUIRED 2.1. Included Materials
2.2. Additional Materials Needed
3. EXPERIMENTAL PROTOCOL 3.1. Assay Buffer Preparation 3.2. Reagent Preparation Prepare this solution in a plastic container, as the reagent may adsorb to glass surfaces. The working solution of OliGreen® reagent must be protected from light with foil or by placing it in the dark to prevent photo degradation. For best results, use the solution within a few hours of preparation. 3.3. Oligonucleotide Standard Curve 3.3.2. For the high-range standard curve, dilute the 100-µg/mL oligonucleotide standard 50-fold in TE assay buffer to make a 2-µg/mL working solution. Dilute this solution according to the examples shown in Table 1. For the low-range standard curve, first dilute the 2-µg/mL oligonucleotide solution 20-fold with TE assay buffer to make a 100 ng/mL oligonucleotide stock solution. Then use this to prepare the dilutions shown in Table 2. Add 100 µL of each standard to separate wells of a 96-well assay plate. It is recommended to obtain duplicates or triplicates of each standard for best results in determining the accuracy of the standard curve. 3.3.3. Add to each well 100 µL of the appropriate aqueous working solution of OliGreen® reagent, as prepared in Section 3.2. 3.3.4. Set up the Modulus™ Microplate Fluorometer as per instructions in the Operating Manual and read the assay plate. 3.3.5. Subtract the fluorescence value of the reagent blank from that of each sample. Use corrected data to generate a standard curve of fluorescence verses oligonucleotide concentrations.
3.4. Sample Analysis 3.4.2. Add to each sample 100 uL of the OliGreen® reagent as prepared in Section 3.2. Incubate for two - five minutes at room temperature, protected from light. 3.4.3. Measure the fluorescence of the samples. Use the same instrument conditions that were used to generate the standard curve (see Section 3.3.). 3.4.4. If the standard curve has been constructed from background-subtracted data (see Section 3.3.), subtract the reagent blank fluorescence reading from that of each sample. 3.4.5. Determine the oligonucleotide concentration of each sample from the standard curve as generated in Section 3.3. 4. REFERENCE 5. RESULTS Sensitivity:
Dynamic Range:
Minimum Detection Limit:
6. CONCLUSION The Modulus™ Microplate Fluorometer offers both superior sensitivity and dynamic range. The Modulus™ Microplate Fluorometer achieves superior performance by use of a dedicated fluorescence detector. The detector is not shared with any other detection modes. The individual Fluorescent Optical Kit of the Modulus™ Microplate Fluorometer uses solid-state optics and a powerful wavelength-matched LED to deliver excellent sensitivity and dynamic range. The modular approach of the Modulus™ Microplate Fluorometer allows for instrument capability expansion as needs in the lab change. Luminescence and/or Absorbance Detection Modules as well as other accessories can be added after initial purchase. 7. WARNINGS AND PRECAUTIONS Caution: No data are currently available addressing the mutagenicity or toxicity of Quant-iT™ OliGreen® reagent. This reagent binds to nucleic acids, and therefore should be treated accordingly 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. It is strongly recommended to use double gloves when handling the DMSO stock solution. As with all nucleic acid reagents, solutions of Quant-iT™ OliGreen® reagent should be poured through activated charcoal before disposal. Incinerate the charcoal to destroy the dye. TRADEMARK For research use only. Not for use in diagnostic procedures. Modulus is a trademark of Turner BioSystems, Inc. All other trademarks are the sole property of their respective owners. TURNER BIOSYSTEMS CONTACT INFORMATION Orders for Turner BioSystems' products may be placed by: Phone: (408)
636-2400 Contact us
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Promega Corporation Sunnyvale Office • Detection Instruments 645 N Mary Avenue • Sunnyvale CA 94085 Phone: 408.636.2400 - Toll Free: 888.636.2401 - Fax: 408.737.7919 |
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