Detection of 6-Dye PowerPlex® System Amplified Fragments

Using the SeqStudio Instrument

These instructions are meant to be used as guidelines and have not been validated at Promega.

Please refer to the technical manual for the specific 6-dye PowerPlex® System you are using for
complete product description and storage conditions.

Materials to Be Supplied by the User

 95°C dry heating block, water bath or thermal cycler

 Crushed ice or ice-water bath
 Centrifuge compatible with 96-well plates
 SeqStudio Consumable Cartridge
 SeqStudio Buffer Container
 MicroAmp® optical 96-well plate
 Aerosol-resistant pipette tips
 Hi-Di™ formamide

Sample Preparation

1. Thaw the Internal Lane Standard WEN ILS500.

Note: Centrifuge tube briefly to bring contents to the bottom, then vortex for 15 seconds
before each use. Do not centrifuge after vortexing, as this may cause the size standard to be
concentrated at the bottom of the tube.

2. Prepare a loading cocktail by combining and mixing Internal Lane Standard 500 and Hi-Di™
formamide as follows:
[(0.5µl ILS 500) x (# samples)] + [(9.5µl Hi-Di™ formamide) x (# samples)]
Note: The volume of internal lane standard used in the loading cocktail can be increased or
decreased to adjust the intensity of the size standard peaks based on laboratory
preferences.

3. Vortex for 10-15 seconds to mix.

4. Pipet 10µl of formamide/internal lane standard mix into each well.

 Detection of 6-Dye PowerPlex® System Amplified Fragments
Using the SeqStudio Instrument

5. Add 1µl of amplified sample (or 1µl of Allelic Ladder Mix) to each well. Cover wells with
appropriate septa.
Note: Instrument detection limits vary; therefore, injection time, injection voltage or the
amount of sample mixed with loading cocktail may need to be adjusted (see Instrument
Preparation below). If the injection time or voltage is reduced, a decreased peak amplitude
threshold for the red channel may be required for proper sizing.

6. Centrifuge plate briefly to remove air bubbles from the wells.

7. Denature samples at 95°C for 3 minutes, then immediately chill on crushed ice or in an ice-
water bath for 3 minutes. Denature samples just prior to loading the instrument.

Creating a Size Standard

1. From the Home screen Select “Settings”. Select “Run Settings”. Select “Size Standard”
scroll to GS600LIZ and select “Copy”.
 Detection of 6-Dye PowerPlex® System Amplified Fragments
Using the SeqStudio Instrument

2. Select “Name” field and rename “Promega ILS 500”.

 Detection of 6-Dye PowerPlex® System Amplified Fragments
Using the SeqStudio Instrument
3. Select all fragment sizes shown below and select “Delete”.

4. Select “Add”, type “65” and then select “Add” (below Add Field). Repeat this step,
adding fragment sizes: 225, 275, 325, 350, 375, 425, 450 and 475 fragment sizes.
 Detection of 6-Dye PowerPlex® System Amplified Fragments
Using the SeqStudio Instrument
5. Verify correct fragment sizes are present. Select “Done” and use arrow key to return to
home screen.

Instrument Preparation

This protocol provides general guidelines for use of Promega 6-dye chemistries after spectral
calibration with a Promega 6-Dye Matrix (DG4900) kit on the Applied Biosystems SeqStudio
Instrument. Some optimization may be required.

1. From the home screen, select “Set up run”.

a. If you’ve used PlateManager software to create your plate layout, navigate to
the plate setup (Cloud, USB, Network Drive, etc).
 Detection of 6-Dye PowerPlex® System Amplified Fragments
Using the SeqStudio Instrument
b. To generate a new plate layout, select “Create new plate setup”.

2. Defining plate properties-

a. In the plate properties screen, define Plate name and Save location. Barcode and
Owner are fields are optional.
 Detection of 6-Dye PowerPlex® System Amplified Fragments
Using the SeqStudio Instrument
b. Under the Applications dropdown menu, select “Fragment Analysis” or “Mixed”
for mixed-use plates (i.e. Fragment and Sequencing).
c. Select “More Options” to check the Plate setup security, analysis settings and file
name convention. Select “Done” to return to Plate Properties.

3. Assigning well contents to plate layout-

a. Navigate to “Plate” in the upper right corner of the screen.
b. Select injection groups containing sample wells and select “edit”.
 Detection of 6-Dye PowerPlex® System Amplified Fragments
Using the SeqStudio Instrument
c. From the Run Module dropdown list, select “FragAnalysis”.

d. From the Size Standard dropdown list select “Promega ILS 500”.
 Detection of 6-Dye PowerPlex® System Amplified Fragments
Using the SeqStudio Instrument
e. From the Dye Set drop down list, select “Promega 6C”.

4. Assigning sample names and types-

a. From the Edit Plate screen, select “Sample Name” field.
 Detection of 6-Dye PowerPlex® System Amplified Fragments
Using the SeqStudio Instrument
b. Edit sample names accordingly.

c. Navigate to “Sample Type” field.

 Detection of 6-Dye PowerPlex® System Amplified Fragments
Using the SeqStudio Instrument
d. Assign sample type accordingly (i.e. Sample, Positive Control, Negative Control or
Allelic Ladder).

e. Select “Done” to close the screen and “Done” to close Plate Editor.
5. Select “Save” to save plate layout for later or select “Start Run” to process plate
immediately.

Generally, the default injection parameters for this run module work well, but run
time, injection time and other instrument settings should be optimized and validated
in your laboratory.