Tagment Genomic DNA

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

• EBLT (Enrichment Bead-Linked Transposomes)
• TB1 (Tagmentation Buffer 1)
• Nuclease-free water
• 8-tube strip
• 96-well PCR plate
• Microseal 'B' adhesive seal
• Pipette tips
– 20 µl
– 200 µl

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 upright so that the beads are always submerged in the buffer.
– Do not use EBLT that has been stored below 2°C.
1. Prepare the following consumables:
• EBLT—Vortex to mix. Do not centrifuge before pipetting.
• TB1—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 10–1000 ng.
2. If DNA volume is < 30 µl, add nuclease-free water to the DNA samples to bring the total volume to 30 µl.
3. Vortex EBLT (yellow cap) for 10 seconds to resuspend. Repeat as necessary.
4. For each sample, 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 to ensure accurate pipetting.

5. Vortex the Tagmentation Master Mix for 10 seconds to resuspend.
6. Divide the Tagmentation Master Mix volume equally into an 8-tube strip.
7. Using a 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.

8. Discard the 8-tube strip after the Tagmentation Master Mix has been dispensed.
9. Using a multichannel pipette set to 40 µl, pipette each sample 10 times to resuspend, and then seal the plate. Alternatively, seal the plate and shake at 1600 rpm for 1 minute.
10. Place on the preprogrammed thermal cycler and run the TAG program.
11. Wait until the TAG program has reached the 10°C hold temperature before removing the plate and proceeding.