Name: Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye (Video) | JoVE Journal
Uploaded: 2010-11-05T17:00:00.000+00:00
Duration: 7 min 44 s
Description: Learn dsDNA quantification using PicoGreen dye and the Hitachi F-7000 Fluorescence Spectrophotometer with microplate reader. This method establishes a linear calibration curve for accurate DNA measurement in biological research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

1. Instrument Setup
<ol>
 <li>Turn on the spectrofluorometer and allow to warm up for 1 hour.</li>
 <li>Using Excel create a file with the Standards and Sample data, as shown in Figure 1 and save it in comma separated values "csv" format. 
 <img src="/files/ftp_upload/2465/2465fig1.jpg" alt="Concentration data table in spreadsheet format for standard and sample mg/ml measurements." data-altupdatedat="1761809329000" data-alt="Figure 1"> 
 Figure 1. Standards and samples data. 
 
 </li>
 <li>Set up fluorometer as follows:
 <ol>
 <li>Click on the <img src="/files/ftp_upload/2465/2465icon1.jpg" alt="Chemical method flowchart icon with test tube and chromatogram, symbolizing lab analysis techniques." data-altupdatedat="1761809329000" data-alt="Icon 1"> button, this will open the Analysis Method window, shown in Fig 2. 
 <img src="/files/ftp_upload/2465/2465fig2.jpg" alt="Photometry method setup on F-7000 FL Spectrophotometer; data entry interface for spectroscopic analysis." data-altupdatedat="1761809329000" data-alt="Figure 2"> 
 Figure 2. Analysis Method Window, General tab. 
 
 Make the following selections:
 <ul>
 <li>In the "Measurement" field, select Photometry from the pull down menu.</li>
 <li>In "Operator" field, enter the appropriate operator's name.</li>
 <li>There is no need to enter information in the "Instrument" field. This is selected automatically by the software.</li>
 <li>The "Sampling" field is inactive when using the microplate reader.</li>
 <li>Enter any comments which you may want included in the report in the 
 "Comments" field.</li>
 <li>In the "Accessory" field, enter information related to the accessories used for this test.</li>
 <li>Note the buttons on the right side of the window.</li>
 <li>The "Load" button allows loading previously saved Analysis Methods.</li>
 <li>The "Save" button allows updating a set of test parameters or Analysis Method previously saved and loaded, using the same name.</li>
 <li>The "Save As" button allows saving a new set of test parameters or Analysis Method under a desired name.</li>
 </ul>
 Now click on the "Quantitation" tab, see Figure 3 and make the following selections. 
 <img src="/files/ftp_upload/2465/2465fig3.jpg" alt="Quantitation method setup for wavelength analysis; parameter inputs for calibration and concentration." data-altupdatedat="1761809329000" data-alt="Figure 3"> 
 Figure 3. Analysis Method Window, Quantitation tab. 
 <ul>
 <li>In the "Quantitation type" field, select Wavelength. This indicates the fluorescence reading will be made using the selected wavelength.</li>
 <li>In the "Calibration type" field, select 1st order.</li>
 <li>In the "Number of wavelengths" field, select 1.</li>
 <li>In the "Concentration unit" field, enter ng/ mL.</li>
 <li>Leave unselected the boxes for "Manual Calibration" and "Force curve through zero".</li>
 <li>In the "Digit after decimal point" field, select 2.</li>
 <li>In the "Lower concentration limit" field, enter 0.</li>
 <li>In the "Upper concentration limit field, enter 10000.</li>
 </ul>
 
 Now click on the "Instrument" tab and make the following selections. 
 <img src="/files/ftp_upload/2465/2465fig4.jpg" alt="Fluorescence data analysis method interface, spectroscopy parameters setup, emission/excitation options." data-altupdatedat="1761809329000" data-alt="Figure 4"> 
 Figure 4. Analysis Method window, Instrument tab 
 <ul>
 <li>In the "Data mode:" field, select Fluorescence.</li>
 <li>The "Chopping speed" field is inactive at this time, it is used only for phosphorescence measurements.</li>
 <li>In the "Wavelength mode" field, select Both WL fixed.</li>
 <li>In the "EX / WL1" field enter 480nm.</li>
 <li>In the "EM / WL1" field enter 520nm.</li>
 <li>All the other fields under EX and EM are inactive at this time.</li>
 <li>In the "EX slit" field select 5.0nm from the pull down menu.</li>
 <li>In the "EM slit" field select 5.0nm from the pull down menu.</li>
 <li>Leave unselected the box in front of the "PMT Voltage 1-1000V" field.</li>
 <li>In the "PMT Voltage" field, select 700V from the pull down menu.</li>
 <li>In the "Auto statistic calc. number" field select 2.</li>
 <li>In the "Replicates" field select 1.</li>
 <li>In the "Integration time" field select 0.1s.</li>
 <li>In the "Delay" field enter or select 0s.</li>
 </ul>
 Click on the "Monitor" tab. 
 <img src="/files/ftp_upload/2465/2465fig5.jpg" alt="Analysis method settings dialog; Y-axis limit in data processing software setup." data-altupdatedat="1761809329000" data-alt="Figure 5"> 
 Figure 5. Analysis Method window, Monitor tab. 
 <ul>
 <li>In the "Y axis", "Max:" field, select 10000</li>
 <li>In the "Y axis", "Min:" field, select 0.</li>
 <li>Select the box to the left of the "Open data processing after data acquisition".</li>
 <li>Leave unselected the box to the left of the "Print report after data acquisition", unless you want a report to be printed automatically after the readings are completed.</li>
 </ul>
 Click on the "Report" tab, see Figure 6. 
 <img src="/files/ftp_upload/2465/2465fig6.jpg" alt="Analysis method settings for report printing; includes data, method, calibration curve options." data-altupdatedat="1761809329000" data-alt="Figure 6"> 
 Figure 6. Analysis Method window, Report Tab. 
 <ul>
 <li>In the "Output" field, select Print Report from the pull down menu. You could also select "Use Microsoft Excel" if you want the data exported to Excel, or "Use Print Generator Sheet" if using the optional add on program Report Generator which will allow generating custom made reports.</li>
 <li>Click the boxes for the items you want to be included in the report.</li>
 <li>To save all the selected parameters in the different tabs of the "Analysis Method" window, click again on the "General" tab and save them under a file name and in a location where you could find them for future use, and click the "OK" button to close this window.</li>
 <li>This completes setting up the instrument test parameters.</li>
 <li>Now we will set up the microplate reader.</li>
 <li>Click on the <img src="/files/ftp_upload/2465/2465icon2.jpg" alt="MPR software icon for data visualization and analysis in scientific research." data-altupdatedat="1761809329000" data-alt="Icon 2"> button, which will bring the MPR Setup Window on top.</li>
 </ul>
 Select wells for the Standards and Samples in the MPR Setup Window/Plate tab, as shown in Figure 7. 
 <img src="/files/ftp_upload/2465/2465fig7.jpg" alt="MPR setup window diagram for microplate analysis, highlighting sample position adjustments." data-altupdatedat="1761809329000" data-alt="Figure 7"> 
 Figure 7. MPR Setup window, Plate tab. 
 <ul>
 <li>Click on the A1 position and drag the mouse to E1, then click on the <img src="/files/ftp_upload/2465/2465icon3.jpg" alt='Button labeled "Standard Set" in a software user interface.' data-altupdatedat="1761809329000" data-alt="Icon 3"> button. This will select the positions to be used for the standards. Those positions will be highlighted in green color.</li>
 <li>Now we need to select positions F1 to H3 for the samples. Click and drag the mouse starting in position F1, to position H3 and then click on the <img src="/files/ftp_upload/2465/2465icon4.jpg" alt='Button labeled "Sample Set" for selection or input choice.' data-altupdatedat="1761809329000" data-alt="Icon 4"> button, this will highlight those positions in yellow color.</li>
 <li>Then repeat the same operation for positions A2 to E3, to select those positions for sample measurements.</li>
 <li>Now, let's load the file Comma Separated Variable (csv) file prepared in advance with the Standards and Sample information.</li>
 </ul>
 Click on the "Well information" tab of the "MPR Setup Window", as shown in Figure 8. 
 <img src="/files/ftp_upload/2465/2465fig8.jpg" alt="MPR setup diagram for sample positioning on 96-well plate, highlighting initial well coordinates." data-altupdatedat="1761809329000" data-alt="Figure 8"> 
 Figure 8. MPR Setup window, Well Information tab. 
 <ul>
 <li>Then click on the <img src="/files/ftp_upload/2465/2465icon5.jpg" alt='UI button labeled "Load Well Information" for database access in petroleum engineering software.' data-altupdatedat="1761809329000" data-alt="Icon 5"> button. A Windows explorer window will open which as shown in Figure 9, which will allow locating the "Well information.csv" file and loading it. 
 <img src="/files/ftp_upload/2465/2465fig9.jpg" alt="MPR setup window; input DNA for fluorescence assay analysis, showing data import from CSV file." data-altupdatedat="1761809329000" data-alt="Figure 9"> 
 Figure 9. Loading well sample information. 
 After loading the "Well information" file, the "Well information" will look as shown in Figure 10 
 <img src="/files/ftp_upload/2465/2465fig10.jpg" alt="Microplate reader setup interface for concentration data in fluorescence assay analysis." data-altupdatedat="1761809329000" data-alt="Figure 10"> 
 Figure 10. Loaded sample well information. 
 </li>
 </ul>
 </li>
 </ol>
 </li>
</ol>
2. Picogreen Assay Sample Preparation
<ol>
 <li>Prepare 1X TE Buffer (10 mM Tris-HCl, 1 mM EDTA) by adding 1 mL of 20 X TE Buffer to 19 mL dI H2O.</li>
 <li>Prepare PicoGreen Reagent Solution by adding 50 μL 200 X PicoGreen Reagent to 9.95 mL 1 X TE Buffer.</li>
 <li>Prepare standards (Lambda dsDNA) by making serial dilutions of stock dsDNA (100 μg/mL). First prepare a solution of 50 μg/mL dsDNA by adding 25 μL of stock dsDNA (100 μg/mL) and 25 μL 1 x TE Buffer. Then prepare the standard solutions by adding the indicated volumes of dsDNA and buffer as listed in the table below:
 <table border="1">
 <tbody>
 <tr>
 <td>Concentration of dsDNA</td>
 <td>Volume of dsDNA</td>
 <td>Volume of Buffer</td>
 </tr>
 <tr>
 <td>1 μg/mL</td>
 <td>20 μL of 50 μg/mL dsDNA</td>
 <td>980 μL 1 X TE Buffer</td>
 </tr>
 <tr>
 <td>0.1 μg/mL</td>
 <td>100 μL of 1 μg/mL dsDNA</td>
 <td>900 μL 1 X TE Buffer</td>
 </tr>
 <tr>
 <td>0.01 μg/mL</td>
 <td>100 μL of 0.1 μg/mL dsDNA</td>
 <td>900 μL 1 X TE Buffer</td>
 </tr>
 <tr>
 <td>0.001 μg/mL</td>
 <td>100 μL of 0.01 μg/mL dsDNA</td>
 <td>900 μL 1 X TE Buffer</td>
 </tr>
 </tbody>
 </table>
 </li>
</ol>
3. Measurement of Standards and Sample Fluorescence
<ol>
 <li>Pipette 150 μL of each standard into wells A1-E1 of a 96 well plate, per Figure 7, and according to the following table:
 <table border="1">
 <tbody>
 <tr>
 <td>Well</td>
 <td>Standard</td>
 </tr>
 <tr>
 <td>A1</td>
 <td>1 μg/mL</td>
 </tr>
 <tr>
 <td>B1</td>
 <td>0.1 μg/mL</td>
 </tr>
 <tr>
 <td>C1</td>
 <td>0.01 μg/mL</td>
 </tr>
 <tr>
 <td>D1</td>
 <td>0.001 μg/mL</td>
 </tr>
 <tr>
 <td>E1</td>
 <td>1 X TE Buffer</td>
 </tr>
 </tbody>
 </table>
 </li>
 <li>Pipette 150 μL unknown samples into wells F1 to H3, shown in Figure 7</li>
 <li>Pour PicoGreen solution prepared in step 1.2 into a trough designed for multi-channel pipette use. Use a multi-channel pipette to deliver 150 μL PicoGreen solution to wells A1-H3. Mix solution and standards gently with pipette.</li>
 <li>Place plate in dark area and wait 2-5 minutes for reaction to develop.</li>
</ol>
4. Representative Results
A good calibration curve is evaluated using the different parameters provided by the F-7000 software upon measurement of the standards and calculation of the calibration curve by the software.
The determination coefficient indicates the goodness of fit of the measured standards and the calculated calibration curve. The closer this value is to "1", the better the fit of the measured value and calibration curve.
If the value is far from "1", the standards must be re-measured, prepared again, or the fitness of the calibration curve changed.
Figure 11 shows an example of a calibration curve with a calculated determination coefficient= 0.99993, obtained for quantification of dsDNA using PicoGreen dye.
<img src="/files/ftp_upload/2465/2465fig11.jpg" alt="Standard calibration graph, first-order linear regression, ng/ml, concentration analysis, R²=0.999." data-altupdatedat="1761809329000" data-alt="Figure 11"> 
 Figure 11. Example of dsDNA calibration curve.

<ol><li>Singer, V. L., Jones, L. J., Yue, S. T., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+PicoGreen+reagent+and+development+of+a+fluorescence-based+solution+assay+for+double-stranded+DNA+quantitation%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.</a> Analytical Biochemistry. 249 (2), 228-238 (1997).</li>
</ol>

Luis A. Moreno and Kendra L. Cox are employed by Hitachi High Technologies America that produces the instrument used in this article.

Careful pipetting during sample preparation, as well as using a stable and sensitive fluorescence spectrophotometer is essential for obtaining good and reproducible results.
In the case of the fluorescence spectrophotometer used for this test, it is suggested using 300 &mu;L final sample volume in the microplate reader. Lower volume decreases the sensitivity, and a larger volume may cause cross contamination between wells.

<table><tbody><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Reagent</td><td></td><td></td><td></td></tr><tr><td>Quant-iT Picogreen dsDNA Assay Kit from Invitrogen</td><td>Reagent</td><td></td><td>P7589</td><td></td></tr><tr><td>Nalge Nunc Fluorescence Microplates p/n 237108</td><td>Supply</td><td></td><td>237108</td><td></td></tr><tr><td>12 Channel Eppendorf Micropipette</td><td>Supply</td><td></td><td>3516677</td><td></td></tr><tr><td>1000 &amp;mu;L Eppendorf Pipette</td><td>Supply</td><td></td><td></td><td></td></tr><tr><td>200 &amp;mu;L Eppendorf Pipette </td><td>Supply</td><td></td><td></td><td></td></tr><tr><td>10 mL serological pipet</td><td>Supply</td><td></td><td></td><td></td></tr><tr><td>Aluminum foil</td><td>Supply</td><td></td><td></td><td></td></tr><tr><td>Pipette tips</td><td>Supply</td><td></td><td></td><td></td></tr><tr><td>Hitachi F-7000 Fluorescence Spectrophotometer with FL Solutions 2.1</td><td>Equipment</td><td></td><td>5J1-0003</td><td></td></tr><tr><td>Microplate Reader Accessory for F-7000</td><td>Equipment</td><td></td><td>5J0-0139</td><td></td></tr></tbody></table>

quantification of dsdna using the hitachi f-7000 fluorescence spectrophotometer and picogreen dye

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

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 &#956;L 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.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

The overall goal of this procedure is to demonstrate the capability of the F 7, 000 fluorescent spectrophotometer with Microplate reader to perform double stranded DNA quantification using Pico green dye. This is accomplished by first preparing the samples and reagents. The second step of the procedure is to set up the instrument.The third step of the procedure is loading samples into the Microplate reader. The final step of the procedure is performing the measurement of standards and samples. Ultimately, results can be obtained that show a linear calibration curve, which could be used for double stranded DNA quantification of unknown samples.Turn on the Hitachi F 7, 000 Spectra Fluorimeter to allow the Xenon lamp to warm up for one hour in Excel. Create a file with the standards and sample data and save it in comma separated values or CSV format. To set up the fluorimeter first, click on the method button that opens the analysis method window.In the measurement field, select photometry from the pull down menu. Now click on the quantitation tab. In the quantitation type field, select wavelength to indicate the fluorescence reading will be made using a specified wavelength.For the calibration type, select first order, and for the number of wavelengths, select one. Enter nanograms per milliliter for the concentration unit. Ensure that manual calibration and force curve through zero boxes are not checked for digit After decimal point, select two, enter zero for lower concentration limit, and 10, 000 for upper concentration limit field.Next, click on the instrument tab and in the data mode field select fluorescence. Verify that the chopping speed field is inactive as it is only used for phosphorescence measurements. In the wavelength mode field, select both WL fixed in the ex WL one field, enter 480 nanometers in the EM WL one field.Enter 520 nanometers. Leave all the other fields under EX and EM as inactive. Set both the ex slit and EM slit to 5.0 nanometers.Using the pull down menus, leave the box in front of the PMT voltage one to 1000 volts field unselected. For the PMT voltage, select 700 volts from the pull down menu. Also, select two for auto statistic calc number one for replicates 0.1 seconds for integration time and zero for delay.Now open the monitor tab and set why axis max field to 10, 000 and why axis min to zero. Then select the box to the left of the open data processing after data acquisition, and if required, the box to the left of the print report after data acquisition for a report that is to be printed automatically on completion of readings. Finally, to save all the selected parameters in the different tabs of the analysis method window, click on the general tab and save under a new file name in a specified location.Click the okay button to close this window. This completes setting up the instrument test parameters. In order to set up the microplate reader, click on the MPR button to bring the MPR setup window to the front.First select wells for the standards. For example, click on the A one position and drag the mouse to E one. Then click on the standard set button.This will select the positions to be used for the standards as highlighted in green color. Then select positions for the samples. Similarly, click and drag the mouse over corresponding positions on the plate, and then click on the sample set button, which will automatically highlight selected positions in yellow.Continue to repeat the same operation for positions of desired sample measurements. To load the standards and sample information onto the comma separated variable file prepared earlier. Click on the well information tab of the MPR setup window.Select the load well information button and a Windows Explorer window will open to identify well information dot csv file. After loading the information, the window should display as a table. In order to prepare a working solution of Pico green reagent, add 50 microliters of 200 X pico green reagent to 9.95 milliliters of TE buffer.For the standard curve, prepare serial dilutions of Lambda double stranded DNA in TE buffer for final concentrations, ranging from one to 100 nanograms per milliliter pipette 150 microliters of each standard into designated wells of a 96 well plate plate. Next pipette 150 microliters of each test sample into corresponding designated wells of the plate. Now using a multi pipette add 150 microliters of Pico green solution to each well and mix the solution and DNA samples.Gently incubate the plate in a dark area for two to five minutes for the reaction to develop. Click on the MPR display screen button to bring the MPR setup window on top of the display and click on the exchange position button. The microplate holding platform will move toward the front of the instrument, open the door of the microplate reader and place the 96 well plate on the holding platform with the A one well toward the front and close the door of the Microplate reader.Click on the initial position button to place position A one of the microplate in the measuring position. Click on the monitor display button to bring the measuring window on top of the display. Click on the measure button to start measuring the standards and unknown samples.A good calibration curve is evaluated using the different parameters provided by the F 7, 000 software. For instance, this ideal calibration curve obtained for quantification of double stranded DNA using Pico green dye has calculated determination coefficient of 0.99993. The high throughput system described is a serial assembly of technologies including Pico Green Dye, a Hitachi F 7, 000 fluorescent spectrophotometer, equipped with Microplate reader and a software program to automate data collection, storage analyses and presentation.The combination of powerful technologies can address the increasing need to accurately analyze small quantities of double stranded.DNA.

quantification-dsdna-using-hitachi-f-7000-fluorescence

Research

JoVE Journal

Biology

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop Microvolume Quantitation of Nucleic Acids

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified λ DNA substrate and a Quantum-dot labeled protein.

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

This study presents a simple, user-friendly ratiometric FRET assay for the detection and quantitative assessment of DNase activity, and demonstrates its application in the analysis of a weak nuclease.

Assessment of DNase Activity by Ratiometric Fluorescence Resonance Energy Transfer

This protocol outlines the design of split hybridization probes utilizing fluorescent light-up aptamer DAP-10-42 as a signal reporter and their application for nucleic acid detection and differentiation between targets with single-nucleotide substitutions.

Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis

This protocol provides detailed operations on sample preparation, data collection, and analysis of DNA intrinsic fluorescence spectra.

Spectroscopic Super-resolution Imaging of DNA Molecules using Intrinsic Contrast

Learn dsDNA quantification using PicoGreen dye and the Hitachi F-7000 Fluorescence Spectrophotometer with microplate reader. This method establishes a linear calibration curve for accurate DNA measurement in biological research.

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Explore More Videos