Tagment Genomic DNA

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This step uses the Enrichment Bead-Linked Transposomes (Enrichment BLT, eBLT) to tagment the input DNA, which is a process that fragments and tags the DNA with adapter sequences.

• eBLT (Enrichment Bead-Linked Transposomes) (yellow cap)
• TB1 (Tagmentation Buffer 1)
• Nuclease-free water
• 96-well PCR plate
• 1.7 ml microcentrifuge tubes
• 8-tube strip
• Microseal 'B' adhesive seal
• Pipette tips
– 200 µl multichannel pipettes

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.

• eBLT:
– Must be stored vertically at temperatures above 2°C.
– Do not use eBLT that has been stored below 2°C.
1. Prepare the following consumables:

Item

Storage

Instructions

eBLT (yellow cap)

2°C to 8° C

Bring to room temperature by incubating at room temperature for 10 minutes. Vortex to mix. Do not centrifuge before pipetting.

TB1

-25°C to ‑15°C

Bring to room temperature. Vortex to mix.

2. Save the following TAG program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 50 µl
• 55°C for 5 minutes
• Hold at 10°C
1. Add 2–30 µl DNA to each well of a 96-well PCR plate so that the total input amount is 50–1000 ng.
• If the DNA volume is < 30 µl, add nuclease-free water to the DNA samples to bring the total volume to 30 µl.
2. Vortex eBLT (yellow cap) vigorously for 10 seconds to resuspend. Repeat as necessary.
3. Combine the following volumes to prepare the Tagmentation Master Mix. Multiply each volume by the number of samples being processed.
• eBLT (11.5 µl)
• TB1 (11.5 µl)

These volumes produce 23 µl Tagmentation Master Mix per sample, which includes extra volume for accurate pipetting.

4. Vortex the Tagmentation Master Mix thoroughly to resuspend.
5. Divide the Tagmentation Master Mix volume equally.

[Optional] Use an 8-tube strip if using an 8-channel multichannel pipette.

6. Transfer 20 µl Tagmentation Master Mix to each well of the plate or tube.

[Optional] Using a 200 µl multichannel pipette, transfer 20 μl Tagmentation Master Mix from the 8-tube strip to each well of the plate containing a sample. Use fresh tips for each sample column.

7. Using a 200 µl multichannel pipette set to 40 μl, pipette 10 times to mix Tagmentation Master Mix and gDNA sample, and then seal the plate. Alternatively, seal the plate and shake at 1600 rpm for 1 minute.
8. Place on the preprogrammed thermal cycler and run the TAG program.
9. Wait until the TAG program has reached the 10°C hold temperature before removing the plate and proceeding.