Scroll down on this page to see all of the steps of the 96-well PCR tutorial, using the "Auxilia" HIV case as an example.

IMPORTANT: Screen shots on this page were taken from Case It v5.03. The red 'Open' button on the 96-well plate is not present in v5.03 (the silver button bar is used instead to open files). If you are using a version prior to 5.03, please go to the home page and upgrade to the latest version of the Case It software.

IMPORTANT: In this software, 96-well PCR is used ONLY to determine viral load for infectious diseases cases such as HIV. Another PCR procedure is used to generate PCR products, such as those used for certain genetic disease cases.

Before going through the 96-well PCR tutorial, go through the ELISA tutorial and the Western blot tutorial.

Once a person has been determined to be HIV+ via the ELISA/Western blot procedures, the 96-well PCR procedure is used to determine viral load over time. We will do this for Auxilia in Case A...

1. Before running the PCR procedure, use the "Clear" menu to clear everything on the Data Screen...

2. If the "ELISA Dotblot 96wPCR" window is not visible, use the button on the Navigation Bar at the bottom of the screen to make it visible.

3. First, click the "Method" button and select "96-well PCR -> Add DNA samples..." as shown below.

4. Next, click the "Open" button and select the "DNA" option...

NOTE: Versions previous to Case It v5.03 have a red 'Open' button next to the 'Method' button, which should not be used because it did not consistently open files. If you are using one of these earlier versions, use the silver button bar instead of the red 'Open' button (or better yet, upgrade to the latest version).

 

5. Click "DNA Auxilia 1 mo.txt", hold down the Shift key, and click "DNA Auxilia 6 mo.txt". This will hilight all three DNA files. Click the "Open" button to open these three files simultaneously...

6. Click the "DNA: Auxilia 1 mo.txt" line in the "Opened & processed" window, then click the first well (A1). The dot in the center of the well will turn purple, indicating it has been selected for loading...

7. Click the "Load" button at the bottom of the window, and the center dot will turn orange, indicating it is loaded.

Repeat this process for the "Auxilia 3 mo." and "Auxilia 6 mo." lines, so that three wells are loaded as shown below. Note that the labels automatically appear on the right.

8. Next, click the "Primer" button, and choose "primers HIV.txt" as shown below...

 
9. All three of the wells will have the same primer, so drag from the upper left to the lower right as shown below to select all three well (or, you could click them one at a time)...

10. The circles for each well turn purple, indicating that they have been selected. Click the "Load" button, and the circles turn orange indicating that this primer has been associated with the three wells, as shown below...

11. Click the "Run" button to run the PCR, and note that in this case all three of the wells turn fluorescent green, indicating a positive result.

12. To determine the viral load for these samples, click the "Options" button and select "Data -> Viral load". Note that the viral load values take the place of the first line for each of the labels, as shown below...