Hitachi F-7000 प्रतिदीप्ति स्पेक्ट्रोफोटोमीटर और PicoGreen डाई का उपयोग dsDNA की मात्रा का ठहराव
Summary
DsDNA आण्विक जांच PicoGreen डाई और Hitachi एफ-7000 प्रतिदीप्ति एक microplate रीडर गौण के साथ सुसज्जित स्पेक्ट्रोफोटोमीटर का उपयोग की मात्रा का ठहराव के प्रदर्शन.
Abstract
Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 μL sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or “csv” format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.
Protocol
Discussion
नमूना तैयार करने के दौरान सावधानी pipetting, के रूप में के रूप में अच्छी तरह से एक स्थिर और संवेदनशील प्रतिदीप्ति स्पेक्ट्रोफोटोमीटर का उपयोग अच्छे और प्रतिलिपि प्रस्तुत करने योग्य परिणाम प्राप्त करने के लिए आवश्यक है.
प्रतिदीप्ति इस परीक्षण के लिए इस्तेमाल किया स्पेक्ट्रोफोटोमीटर के मामले में, यह 300 μL microplate रीडर में अंतिम नमूना मात्रा का उपयोग करने के लिए सुझाव दिया है. कम मात्रा में संवेदनशीलता कम हो जाती है, और एक बड़ी मात्रा में कुओं के बीच पार संदूषण के कारण हो सकता है.
Disclosures
The authors have nothing to disclose.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Distilled H2O | Reagent | |||
| Quant-iT Picogreen dsDNA Assay Kit from Invitrogen | Reagent | P7589 | ||
| Nalge Nunc Fluorescence Microplates p/n 237108 | Supply | 237108 | ||
| 12 Channel Eppendorf Micropipette | Supply | 3516677 | ||
| 1000 μL Eppendorf Pipette | Supply | |||
| 200 μL Eppendorf Pipette | Supply | |||
| 10 mL serological pipet | Supply | |||
| Aluminum foil | Supply | |||
| Pipette tips | Supply | |||
| Hitachi F-7000 Fluorescence Spectrophotometer with FL Solutions 2.1 | Equipment | 5J1-0003 | ||
| Microplate Reader Accessory for F-7000 | Equipment | 5J0-0139 |
References
- Singer, V. L., Jones, L. J., Yue, S. T., Haugland, R. P. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Analytical Biochemistry. 249 (2), 228-238 (1997).
