Tagment PCR Amplicons

This step uses EBLTS to tagment the influenza A/B virus whole genome RT-PCR amplicons. The tagmentation process fragments and tags the amplicons with adapter sequences.

• EBLTS (Enrichment BLT)
• TB1 (Tagmentation Buffer 1)
• Nuclease-free water
• 1.7 ml tube
• 2 ml tube
• 96-well PCR plate
• Microseal 'B' adhesive seal
• EBLTS
– Store upright at temperatures above 2°C. Make sure beads are always submerged in the buffer.
– If beads are adhered to the side or top of the wells, centrifuge at 500 × g for 1 minute, and then pipette to resuspend.
1. Prepare the following consumables:
• EBLTS—Vortex to mix.
• TB1—Vortex to mix.
2. If the TAG1 plate was stored frozen, prepare as follows:
a. Thaw at room temperature.
b. Check seals, and then shake at 1600 rpm for 1 minute.
c. Centrifuge at 1000 x g for 1 minute.
3. Save the following IMAP-Flu TAG program on the thermal cycler:
• Choose the preheat lid option at 105°C
• Set the reaction volume to 50 µl
• 55°C for 5 minutes
• Hold at 10°C
1. In a 2 ml tube, combine the following volumes to prepare Tagmentation Master Mix. Multiply each volume by the number of samples. Reagent overage is included to account for small pipetting errors.
• TB1 (12 µl)
• EBLTS (4 µl)
• Nuclease-free water (20 µl)
2. Add 30 µl Tagmentation master mix to each well of the TAG1 plate.
3. Seal and shake at 1600 rpm for 1 minute.
4. Place on the preprogrammed thermal cycler and run the IMAP-Flu TAG program.