GMT Tutorial Exercise 22 - Workflow 1 - Step 7

Using the macro - the "Primer Dilution Table" worksheet

• This screenshot shows the Primer Dilution Table worksheet of the "Automated Designer for Marker Screening".
• Enter experimental data following the instructions on this worksheet. See below for further explanations.
• Print or save the primer dilution table, using the Print Preview or the Save Table as New File buttons. This table is required to set up PCR assays in the lab.

(1) Enter the volume of diluted primer required for each marker and press Calculate.

• For each PCR reaction, it is assumed that 3 µL of a mastermix, containing all PCR components with the exception of the locus-specific primers (i.e. including genomic DNA), are mixed with 3 µL of a diluted primer mix, containing both the forward and reverse locus-specific primer.
• The value to be entered in this field is calculated as:

¹Extra volume:
This is required to make up for pipetting losses and for the "dead volume" below which air is likely to be picked up during pipetting. The extra volume depends on the plastic ware and the pipetting method used, e.g. the dead volume in robotic pipetting is likely to be bigger than when using a single channel manual pipette. You will have to estimate the necessary extra volume based on your experience with your pipetting method.

Example:
If 6 DNA samples are to be screened with each marker, and the extra volume is estimated to be ca. 10 µL, the required volume of diluted primer is:

• Once the required volume of diluted primer has been entered into the macro, hit the Calculate button.
• A primer dilution table is automatically calculated, based on:
  1. The optimal nanomolar final concentrations determined for locus-specific primers in the multiplex-ready^TM assay (listed under heading "LSPopt" in the primer dilution table).
  2. The assumption that all locus-specific primers are available as a mix of forward and reverse primer, with both primers at a concentration of 0.4 µM (this primer mix is abbreviated as "0.4 µM LSP" in primer dilution table).
  3. The assumption that 3 µL diluted primer are mixed with 3 µL PCR master mix, resulting in an additional 2-fold dilution.
• Do not forget to print or save the primer dilution table, using the Print Preview or the Save Table as New File buttons. This table will be used when setting up PCR assays in the lab.
• If using the Save option, an output file is saved in the folder where the Automated Designer for Marker Screening is found. The macro creates a file name by appending _DilTable.xls to the experiment name (as entered on the Data Entry worksheet).
• When finished, click on Next Sheet to reach the [next step] of this workflow.