Make PGx (PGx only)

This step adds the DNA samples and reagents to the plates to prepare them for PCR amplification.

• MA1
• PGM (PGx Master Mix)
• PGP (PGx Primer Pool)
• 96-well 200 μl skirted PCR plate
• DNA plate (20 ng/μl per well)
• Microseal 'B' adhesive seals
1. Prepare the tubes of MA1, PGM, and PGP based on the number of sample plates.

Number of plates

MA1

PGM

PGP

0.5

1

1

1

1

2

1

1

2

4

2

2

3

6

3

3

4

8

4

4
2. Prepare the following consumables:

Reagent

Storage

Instructions

MA1

Room temperature

Vortex or invert 10 times to mix. Centrifuge at 280 x g for 1 minute.

PGM

-25°C to -15°C

Thaw at room temperature. Vortex or invert 10 times to mix. Centrifuge at 280 × g for 1 minute. Keep on ice until use.

PGP

-25°C to -15°C

Thaw at room temperature. Vortex or invert 10 times to mix. Centrifuge at 280 × g for 1 minute.
3. Thaw samples to room temperature. 
4. In the post-amp area, save the following PCR program on the thermal cycler:
• Set the reaction volume to 35 μl and the lid temperature to 105°C
• 98°C for 30 seconds
• 11 cycles of:
• 98°C for 30 seconds
• 76°C for 30 seconds
• 72°C for 20 seconds
• 21 cycles of:
• 98°C for 30 seconds
• 66°C for 30 seconds
• 72°C for 20 seconds
• 72°C for 5 minutes
• Hold indefinitely at 12°C
5. Apply a PGx barcode label to a new PCR plate.
1. Add 10 µl MA1 to each well of the PGx plate.
2. Add 10 µl PGP to each well of the PGx plate.
3. Add 10 µl PGM to each well of the PGx plate.
4. Transfer 5 µl DNA sample from the DNA plate to the corresponding wells of the PGx plate.
5. Seal the PGx plate. Make sure each well is tightly sealed with no bubbles or gaps in the film.
6. Vortex at 1600 rpm for 15 seconds, and then centrifuge at 280 × g for 1 minute.
7. Perform the remaining protocol steps for the PGx plate in the post-amplification area.