GMT Tutorial Exercise 21 - Workflow 1 - Step 7

Using the macro - the PCR Master Mixes worksheet

• This screenshot shows the PCR Master Mixes worksheet of the "Automated Designer for Whole Genome Scan".
• Enter experimental data following the instructions on this worksheet. See below for further explanations.
• Use the Print Preview button to print out the PCR master mixes for setting up PCR assays in the lab.

1. "Excess volume of master mix" (cell reference J248 on the Excel spreadsheet)
   • Enter the percentage of extra master mix volume to be prepared, the default setting is "10%". This extra volume is required to allow for losses and for the "dead volume" below which air is likely to be taken up during pipetting. Both of these depend on the plasticware and the pipetting method used, so you may want to choose a different percentage, based on your experience with dispensing PCR master mixes.
   • Notes:
     • The PCR master mixes contain all the PCR reagents (including genomic DNA), except the "locus-specific" primers (LSP).
     • For each DNA sample, 4 different master mixes have to be prepared, each with an MRT^TM tag-specific primer labelled with a different fluorescent dye (VIC, FAM, NED and PET, indicated above each master mix).
     • The size of each master mix will vary according to the total number of markers that will be assayed in a particular dye detection channel for each DNA sample.
     • To set up a PCR assay, 3 µL of PCR master mix will have to be mixed with 3 µL of the diluted "locus-specific" primer (prepared according to the instructions on the Primer Dilution Plate Layout worksheet, and combined according to the layout specified on the PCR Setup worksheet).
2. "Enter concentration of DNA sample"
   • For each of the DNA samples, enter the concentration in ng µL^-1.
   • DNA concentrations have to be at least 50 ng µL^-1.
3. Click the Calculate Mixes button, the master mix information is calculated.
4. Click on Print Preview to print a copy for use in the lab.