Index PCR

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In this step, library fragments are amplified using primers that add index sequences for sample multiplexing. The resulting product contains the complete library of cfDNA fragments flanked by index sequences and adapters required for cluster generation.

• 96-well PCR plate, LoBind
• Microseal 'B' adhesive seals
1. Label a new 96-well LoBind PCR plate ALS.
2. In the post amplification area, save the following I‑PCR program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 50 µl
• 98°C for 30 seconds
• 15 cycles of:
• 98°C for 10 seconds
• 60°C for 30 seconds
• 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 10°C
1. Transfer the LP plate to the post-amplification area.
2. Centrifuge briefly.
3. Place the LP plate on the preprogrammed thermal cycler and run the I-PCR program.
4. When the program reaches the 10°C hold, remove from the thermal cycler and centrifuge briefly.
5. Place on the bar magnetic stand and wait until the liquid is clear (2 minutes).
6. Without disturbing the beads, transfer 45 μl supernatant from each well of the LP plate to the corresponding well of the ALS plate.

Minor bead carry-over might occur and does not inhibit downstream steps.

7. Discard the LP plate.

SAFE STOPPING POINT

If you are stopping, apply Microseal 'B' to the ALS plate, and briefly centrifuge at 280 × g. Store at 2°C to 8°C overnight, or at ‑25°C to ‑15°C for up to 30 days.