QPCR - Lin Lab

Before the run:

1. Click on “Shortcut to Sybr plate” icon.
2. Click on “Instrument” tab and change volume of rxn. to 20uL. With new primers, may want to run the dissociation step to see if one peak (or more) is generated by the primers. This step adds about an extra hour to the run time. Can eliminate this step for primers which have already been analyzed in this way.
3. Go to File menu and then Save As. Go up one folder to ABI folder then to the Results folder, then save in the Saltiel folder (in my folder).
4. Close the program (Shortcut to Sybr plate window).
5. Reopen the program through the 7300 System Software icon. Open the new saved file and the 7300 system should then initialize.
6. Under the Instrument tab, hit “Start.” Note: It takes a few minutes for the run to begin after “Start” is hit. Sometimes the run time is incorrectly calculated by the software.

After the run:

(Steps 1 and 2 are only necessary if the file is not examined immediately after the run.)

1. Click on the “Shortcut to Sybr plate” icon.
2. Go to File menu then Open. Go up one folder to the Results folder and open the Saltiel folder. Then open my folder to get to the file to open.
3. Hit the “Results” tab then the “Amplification” plot to review the amplication curves. Highlight cells on the plate grid to see the curves associated with the cells.
4. Check the Dissociation Curve to see if there is one peak for particular primer pairs. Again, highlight cells on the plate grid to see the curves associated with the cells.
5. Go to “Amplification Plot” or go to the Analysis menu. Select Auto CT then select Analyze (also under the Analysis menu). Can also set the threshold manually. Don’t worry about the baseline as long as the end cycle isn’t set at too high a cycle number and might interfere with the analysis. The scale on the Y-axis can be adjusted on the graph to make it easier to select the threshold. Click on the Y-axis to open a pop-up window to manually set the Y-axis.
6. Hit “Report” and highlight the cells used. Go to “Export” then “Results” under the File menu. There will be a .csv file which Excel can read.
7. Export the report in my folder inside the “Results” folder. Exit the software and save changes to “Absolute Quantification.”
8. To transfer the USB drive, insert the device then go to My Computer to open the USB window (i.e. the removeable disk window) if it does not open automatically. Then go to “Shortcut to results”icon to open that window. Drag and drop the .sds (raw data) and .csv (Excel spreadsheet) files into the USB window. Right click on the green arrow hardware button at the bottom right of the screen. Then hit the U3 icon next to the green arrow to open the U3 window. Hit the Eject button to safely remove the USB drive.

PCR Program:

Stage 1	Stage 2	colspan = "4"| Dissociation
Rep 1	Rep 1	colspan = "4"| Stage 4
		colspan = "4"| Rep 1
50C	95C	95C	60C	95C	60C	95C
2 min	10 min	15 sec	1 min	15 sec	30 sec	15 sec

Protocol written for 96-well Real-time PCR machine in Jiandie Lin lab on 10/4/2006.