GMT Tutorial Exercise 23 - Workflow 2 - Step 8

Using the macro - the PCR Master Mixes worksheet

• The screenshot below shows the PCR Master Mixes worksheet of the "Automated Designer for Genetic Analysis, University of Adelaide Special Edition".
• Fill in experimental details, following the instructions on this worksheet, see also further explanations below.
• Print out or save the PCR set up instructions on this worksheet. These will be required when preparing PCR assays.
• Click on Generate PANEL File to save a panel file. This will be required to analyse electrotraces with GeneMapper, see also the next step of this workflow for more details on the PANEL file.

(1) Enter the number of PCR reactions required

• The number of PCR reactions is the number of samples to be analysed with each marker, plus extra master mix aliquots to account for the amount of PCR master mix typically lost during pipetting. As these losses are influenced by the plasticware and pipetting method used, they have to be estimated based on your experience with your set up.
• When estimating the number of extra PCR reactions to be made up, also consider the total volume of master mix that is added to each assay, see instruction (2) below.

(2) Select the volume of master mix and gDNA to be used in each reaction

• Use the drop-down box next to instruction (2) to choose one of the following available options for combining PCR master mix and genomic (i.e. template) DNA ("gDNA"):
  1. 3 µL master mix + 3 µL gDNA
  2. 4 µL master mix + 2 µL gDNA
  3. 4 µL master mix + 3 µL gDNA

(3) Print out this experimental design by clicking on the Printer icon on the toolbar

• The printout generated following this instruction is going to be used when preparing PCR assays in the lab.
• The example printout below shows how PCR information is recorded:

• In this example, 12 different PCR master mixes will have to be prepared, note the first column entitled Master Mix in the table on the left-hand side.
• The general recipe for the preparation of these PCR master mixes is recorded in the table at the lower right-hand side, entitled 250x Master Mix (in the example, sufficient master mix for 250 aliquots of 3 µL each is prepared).
• The dye colour of the "dye-labelled tagF primer" to be chosen for a given master mix is recorded in the second column of the table on the left-hand side, entitled tagF dye.

• The volume (in µL) of 4 µM "locus-specific primer mix" (i.e. a solution containing both the forward and reverse locus-specific primers for a marker, both at a concentration of 4 µM) to be added to a given master mix is recorded in the 5th column of the table on the left-hand side, entitled vol 4 µM LSP.
• The volume (in µL) of PCR-grade water to be added to a given master mix is recorded in the sixth column of the table on the left-hand side, entitled vol water.

(4) Click the Generate PANEL File button

• Click yes at all following prompts.
• This will open the PANEL file worksheet, please go to the next step of this workflow for details.

The Save PCR Master Mix Worksheet button

• Use this to save an electronic copy of the PCR master mix set up sheet.

The Next Page button

• By clicking this button, you will go straight to the DNA Samples worksheet of this macro.