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.
