GMT Tutorial Exercise 22 - Workflow 1 - Step 9

Using the macro - the "PCR Setup" worksheet

• The PCR Setup worksheet allows setting the preferred plate layout on a 384-well PCR plate, while ensuring this is done in a way that allows robotic or manual multichannel pipetting, and that markers belonging to the same BINNER panel can be easily combined after the PCR step:
  The default PCR configuration for marker screening requires that each row of 24 wells on the PCR plates contain a single DNA sample and four marker panels, one for each dye detection channel on the ABI3730. This configuration permits the post-PCR pooling of each row to a single well, which is then prepared for electrophoretic analysis.
• Please follow the instructions on the worksheet, see also further explanations below.

Enter Capillary Codes / Enter DNA Sample

1. "Use information on which "Capillary Code" stands for which markers and..."
   The first table on this Excel worksheet (row references 6-29) lists all the markers on a row that will be analysed together in a capillary on the ABI3730. Each of these capillaries is given a Capillary Code, e.g. "A", "B", "C", ... , shown in the first column of the table. (In the screenshot, markers that will be analysed in capillary "C" are highlighted.)
2. "...which "DNA Sample Code" stands for which sample..."
   DNA Sample Codes are shown before a grey background (rows 32-34 on the Excel spreadsheet). In the example shown in the screenshot, "1" stands for DNA sample "1.Sunco", "2" stands for DNA sample "2.Tasman", etc.
3. "...to enter your preferred PCR plate layout"
   Below the DNA Sample Codes overview, several 384-well PCR plates are drawn with row and column references. Above each of these plate images, a PCR plate number is displayed (e.g. "384-well PCR plate 1"). Follow these instructions to enter your preferred PCR layout:
   • To the left of each PCR plate image, grey column headers instruct the user to Enter Capillary Codes and Enter DNA Sample for each of the rows belonging to a 384-well PCR plate, with further instructions appearing in "hover boxes" (see screenshot below). Use these fields to assign which DNA sample is to be mixed with which "locus-specific primers" in each row of a 384-well PCR plate.
     • DNA samples are referred to by their DNA Sample Code.
     • "Locus-specific primers" are referred to by their Capillary Code.
     • It can require more than one PCR plate to assign a row to every possible capillary / DNA sample combination.
     • It is not possible to assign markers in another combination or order than that shown for each capillary in the table at the top of this worksheet. This would change the "panels" created by BINNER and would lead to overlapping allele size ranges between markers.
       
   • When finished, click the Update PCR Layout button. As a result, each well of the PCR plate images will be "filled" with the name of the "locus-specific" primer and with the "dye colour" of the PCR master mix that will have to be combined in that well, see also PCR set up instructions. The fluorescent dyes and "their" colours are:

   VIC: "green"

   FAM: "blue"

   NED: "yellow"

   PET: "red"

4. Save or print the PCR plate layout for your records
   Click on the Save this worksheet as a new Microsoft Excel file button to save an electronic copy of the PCR plate layout.
   Alternatively, click on Print Preview for Capillary Configuration and on Print Preview for 384-well PCR Plate Setup to generate printouts for use in the laboratory.