Normalize Libraries

This step normalizes the quantity of each library made with Nextera XT Index v2 or Nextera XT Index Kits to ensure more equal library representation in the pooled library.

Do not follow the normalization protocol and instead use Dilute Libraries to the Starting Concentration for manual normalization:

• If you are using IDT for Illumina Nextera UD Indexes.
• If the final library yield is < 10 nM.
• If your sequencing system uses onboard denaturation.
• LNA1 (Library Normalization Additives 1)
• LNB1 (Library Normalization Beads 1)
• LNW1 (Library Normalization Wash 1)
• LNS1 (Library Normalization Storage Buffer 1)
• 0.1 N NaOH (fewer than 7 days old) (3 ml per 96 samples)
• 96-well 0.8 ml polypropylene deep-well storage plate (MIDI plate)
• 96-well PCR plate
• 15 ml conical tube
• Microseal 'B' adhesive seals

About Reagents

• Vortex LNA1 vigorously to make sure that all precipitates have dissolved. Inspect in front of a light.
• Vortex LNB1 vigorously, with intermittent inversion (at least 1 minute). Repeat until all beads are resuspended and no beads are present at the bottom of the tube when it is inverted.
• Always use a wide-bore pipette tip for LNA1.
• Mix only the required amounts of LNA1 and LNB1 for the current experiment. Store the remaining LNA1 and LNB1 separately at the recommended temperatures.
• Aspirate and dispense beads slowly due to the viscosity of the solution.

This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. 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.

Prepare the following consumables:

Item

Storage

Instructions

LNA1

‑25°C to ‑15°C

Prepare under a fume hood.

Bring to room temperature. Use a 20°C to 25°C water bath as needed.

LNB1

2°C to 8°C

Bring to room temperature. Use a 20°C to 25°C water bath as needed.

LNW1

2°C to 8°C

Bring to room temperature. Use a 20°C to 25°C water bath as needed.

LNS1

Room temperature

Keep at room temperature.
1. Transfer 20 µl supernatant from each well of the PCR plate to the corresponding well of a new MIDI plate.
2. Combine the following volumes in a 15 ml conical tube to prepare the LN master mix. Multiply each volume by the number of samples being processed.
• LNA1 (46 µl)
• LNB1 (8 µl)

Reagent overage is included in the volume to ensure accurate pipetting.

3. Pipette 10 times to mix.
4. Pour the LN master mix into a trough.
5. Use a 200 µl multichannel pipette to transfer 45 µl LN master mix to each well.
6. Seal the plate, and then use a plate shaker at 1800 rpm for 30 minutes.
7. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
8. Without disturbing the beads, remove and discard all supernatant.
9. Wash two times with 45 µl LNW1 as follows.
a. Add 45 µl LNW1 to each well.
b. Seal the plate, and then use a plate shaker at 1800 rpm for 5 minutes.
c. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
d. Without disturbing the beads, remove and discard all supernatant.
10. Add 30 µl 0.1 N NaOH to each well.
11. Seal the plate, and then use a plate shaker at 1800 rpm for 5 minutes.
12. Add 30 µl LNS1 to each well of a new 96-well PCR plate labeled SGP.
13. After the 5 minute elution completes, make sure that all samples in the MIDI plate are resuspended.

If they are not, resuspend as follows.

a. Pipette 10 times to mix or lightly tap the sample plate on the bench.
b. Seal the plate, and then use a plate shaker at 1800 rpm for 5 minutes.
14. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
15. Transfer 30 µl supernatant from each well of the MIDI plate to the corresponding well of the SGP plate.
16. Seal the sample plate, and then centrifuge at 1000 × g for 1 minute.

At this point, the libraries are single-stranded DNA, which resolves poorly on an agarose gel or Bioanalyzer chip. For quality control, use the double-stranded DNA saved from step 17 of the cleanup procedure.

SAFE STOPPING POINT

If you are stopping, seal the plate with Microseal 'B' adhesive seal or Microseal 'F' foil seal and store at ‑25°C to ‑15°C for up to 7 days.