Scroll down on this page to see all of the steps of the Multiplex PCR tutorial, using the "Salmonella" case as an example. With Multiplex PCR, more than one PCR product can be generated from the same sample. With Case It v5.03, any time you have more than one primer set in a primer file, the program automatically runs Multiplex PCR [primer set = one forward and one reverse primer]. For this example, the primer file has two primer sets, as you will see as you go through this tutorial.
1. Click the 'DNA' button on the silver button bar and open the Salmonella folder.
|Note: The Salmonella case folder is a separate download - see the download link at the Case It home page.|
Click on the first line in the Salmonella case folder, hold down the shift key, and click on the fourth line to hilight all four lines. Then click the 'Open' button to simultaneously open these four files.
|Note: It may take a few seconds to open the the Salmonella DNA samples- they are very large (over 4.5 million bp each).|
2. Next, click the 'Primer' button on the silver button bar and open the primers file in the Salmonella case folder.
3. You can see the two primer sets in this file by scrolling in the field in the upper left-hand corner of the Data Screen, as shown below.
4a. We could run Multiplex PCR on each DNA sample individually, but it is more efficient to run all four samples simultaneously.
To do this, first go to 'Opened & processed' window and hilight all four DNA lines (click the first line, HOLD DOWN THE SHIFT KEY, and click the fourth line).
4b. CONTINUING TO HOLD DOWN THE SHIFT KEY, click the 'Run' button as shown immediately below...
4c. ...and PCR products are generated for all four files, as shown in the 'Opened & processed' window.
|Note: The above procedure for running PCR on multiple samples works even if you are not doing Multiplex PCR (that is, it works regardless of the number of primer sets per primer file).|
5a. Click the first PCR product line in the 'Opened & processed' window (below left), then click the 'Load' button (below right) to load the first well...
5b. Next, click the second line in the 'Opened & processed' window, click the second well button to select it, then click the 'Load' button to load the second well...
5c. Repeat this procedure for the third and fourth lines, so that all four wells are loaded with the respective PCR products...
6a. After the wells are loaded, use the 'Run' menu and select the first option to run the gel.
6b. Note that the fragments are barely visible (in reality, they wouldn't be visible at all at this point). To make them visible, stain them by clicking the purple button below the gel. Note that there are two fragments in lane 3, meaning that two different PCR products were generated for the 'DNA S_typhimurium' sample, one for each of the primer sets in the primer file. For the other three lanes, only one of the two primer sets resulted in a PCR product.
6c. You could also click the orange button to see the fragments as they would appeara under UV light, or click the black button to see how the gel would appear after it has been photographed. Although not shown here, you could also click the white button to make the fragments completely invisible.
Click the 'Next' button to continue, or click the 'Home' button to go back to the Case It home page...