Index PCR

This step amplifies library fragments and adds index sequences for multiplexing and sequencing.

• Silicone mat for PCR microplate
• The following IPPAS cleanup consumables:
– Bleach disinfectant
– Deionized water

Determine the appropriate disinfectant for IPPAS cleaning procedures, in accordance with your applicable regional, national, and local laws and regulations.

1. Transfer the PCR plate to a plate shaker and shake at 1500 rpm for 1 minute to resuspend.
2. If splashing occurs, centrifuge at 280 × g for 15 seconds to make sure that no liquid is adhered to the plate seal.
3. Visually inspect the plate wells. If bead pellets have reformed, repeat steps 1–2.
4. Place the PCR plate on the preprogrammed thermal cycler and cover with a silicone mat.
5. Run the Index PCR program.
6. When the program has completed, remove the PCR output plate from the thermal cycler.

SAFE STOPPING POINT

If you are stopping, store the sealed PCR plate at ‐25°C to ‐15°C for up to 30 days.

After Index PCR, complete all subsequent steps in the post-PCR area.

This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Ventilation should be appropriate for handling of hazardous materials in reagents. Wear protective equipment, including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle used reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety information, refer to the SDS at support.illumina.com/sds.html.

Before beginning this procedure, review the IPPAS safety and compliance information and safety hazards. Refer to the Illumina Protein Prep Automation System Product Documentation.

1. Remove the index plate from the instrument deck.
2. Remove the remaining consumables and tips from the instrument deck.
3. Save the remaining tips and restack them in columns of eight wafers per stack.

Avoid touching the tips with your gloves. Handle the tips by the black wafer to prevent contamination.

4. Remove the biohazard liquid waste wafer from the liquid waste trough.
5. Decontaminate the wafer as follows.
a. Spray with or immerse in bleach disinfectant. Refer to the manufacturer instructions for contact time.
b. Rinse with deionized water.
c. Leave to air dry.
6. Rinse the liquid waste trough with deionized water.
7. Place the dry wafer back on the liquid waste trough.
8. Discard the biohazard waste and used deionized water according to applicable standards for chemical waste for your region.
9. If spillage has occurred, wipe down the deck and ring magnet as follows.
• Wipe down the deck with a bleach disinfectant. Wipe down the ring magnet with 70% EtOH.

For detailed deck and magnet cleaning procedures, refer to the Illumina Protein Prep Automation System Product Documentation.

• Discard the waste according to applicable standards for chemical waste for your region.
10. Select the X in the upper-right corner of the screen to close ILASS.
11. Use the Windows Start menu shutdown procedure to shut down the IPPAS computer.

The IPPAS instrument also shuts down and the lights turn off.

Illumina recommends shutting down the IPPAS computer at the end of each day.