HOW TO SET UP A Q24 RUN:

Q24 needs:
 Shaker up to 1400 rpm
 Heating block to 80 degrees with solid block
 Sepharose Beads
 Vacuum prep tool and “iron” (from Pyro)
 Start-up package, filter tool (Pyro plates, filter tips, troughs, cartridges,
5x24 kit, Binding Buffer, Annealing Buffer, Denaturation Solution, 1X
Wash Buffer)
 70% Ethanol
 PCR products (1-2 assays designed by Pyro tech rep) and seq. primers
 Non-skirted PCR plates or strip tubes with caps or sealing film
NOTE: There are 3 controls that you should run the first time you run each assay:
 template alone – this sample will prep like a regular sample, but no
sequencing primer should be added at the end
 seq. primer alone – just before putting the plate into the instrument, add
seq. primer with annealing buffer (no template control)
 neg. PCR – this is a PCR that is run with water instead of DNA and serves
as a control for the biot. Primer as well as contamination in the PCR
 For multiplex assays, each template should be run with the seq. primer
from the other template, both templates should be run together with no
seq. primer and both seq. primers together with no template
Double click on PyroMark Q24 Icon on the Computer
Note: Both AQ and SNP Assays will be set up as AQ assays in the software. Results can
be viewed as genotypes in the report.
Assay Entry:
1. In the shortcut browser, right-click the folder you want to place the assay file in
and select New Assay and then Choose AQ Assay or CpG Assay.
2. Type in the File name (name of your SNP) and press Enter.
3. Fill in Sequence to Analyze (sequence IMMEDIATELY following seq. primer)
 SNP is entered with a slash or IUPAC code, deletion entered in brackets.
(multiple SNPs, tri & tetra-allelic SNPs, multiple-base deletion also possible)
CpG sites are entered as YG.
4. Click on Dispensation order
 Software will automatically select dispensation order based on Seq. to
Analyze, automatically injecting negative controls as first dispensation
and first dispensation following the SNP – NOTE: in the case of
homopolymer, (C/TTT), software will use 3 informative peaks.
 Below the dispensation order, the software will show the theoretical
outcomes for your mutation analysis.
 Optional: Name each variable position.


If creating a CpG assay, define custom methylation ranges (optional)
If creating a CpG assay, add bisulfite treatment controls by left-clicking a
bold orange T in the histogram. Preferably choose a bisulfite control in the
beginning of the sequence, and one that is not part of a homopolymer. Be
sure that the control you choose was a C in the original sequence.
5. Save the Assay
Runs (Setup):
1. In the shortcut browser, right-click the folder you want to place the run file in
and select New run from the context menu.
2. Enter the file name and press the Enter key.
3. Select Instrument Method
 PyroMark Q24 Method 001, Rev. A should be selected
4. Fill in the Plate ID, Barcode, and lot numbers for Reagent ID if desired
 These fields are optional
5. Drag the Assay in the shortcut browser to a well or a selection of wells.
 NOTE: It is possible to run 24 different assays on one plate!
6. Each well is divided into three sections. The Assay ID will be in the top
section. Sample ID goes into the middle section, and any Sample Notes can be
entered in the bottom section. To enter a sample ID or note, select the
appropriate section and enter the text.
 Instructions for importing sample IDs can be found under the Help section
of the software-under Run Setup, Define Sample ID and Note Externally.
 NOTE: It is not necessary to import a panel of sample IDs more than one
time. If running a plate with the same sample IDs multiple times, rightclick on a previously saved run, and choose “Copy and Re-Run”.
7. Click the Save button on the toolbar.
8. Print a list of required volumes of reagents and the plate setup; select Pre Run
Information from the Tools menu and, when the report appears, click the
printer icon.
9. Close the run file and copy it to a USB memory stick.
Running the instrument:
1. Prepare your plate and insert it into the instrument.
2. Plug the USB memory stick (containing the run file) into the USB port at the
front of the instrument. Leave it mounted until the run is finished.
3. Select Run in the instruments main menu (using the up and down buttons)
and press OK.
4. Select the run file.
5. Press Select to start the run.
6. When the instrument confirms that the run file has been saved to the USB
memory stick, press Close.
7. Remove the memory stick from the instrument
Runs (Analysis) –
1. Move the processed run file from the USB memory stick to the computer
running PyroMark Q24 Software.
2. Double click on run in the shortcut menu.
3. On the Overview tab, choose AQ or CpG, then click the Blue Play button
and choose Analyze all Wells.
 Each well will be divided into three segments. You can choose what
information you want to see in those segments on the right hand side:
i. Assay
ii. Sample ID
iii. Quality score – each well will be given a quality score: blue =
pass (99% accurate), yellow = check, a genotype was given but
requires human approval, red = fail, usually results from a failed
PCR
iv. Note
v. Mean % (CpG only)-Average of all methylation sites
vi. Ranges (CpG only)-shows the pre-defined methylation level at
each site.
 Well Information – contains reason for well not passing (check or fail)
 Pyrograms – Analysis results are displayed above the variable positions.
i. Right-click on the Pyrogram and choose Display Histograms to
overlay the theoretical histogram for the assay.
ii. Reference peaks - (can be viewed by right click on pyrogram) –
peak can be eliminated as reference peak if known to have
background
iii. Peak heights – point at peak on pyrogram to view peak height
iv. Click on results to edit quality score
v. To compare two Pyrograms, right-click on the second Pyrogram,
and choose “Open in Lower Area”.
Report – To generate a report, select the desired report from the Reports menu. For more
information about each report, see the Help section of the software.