Tagment PCR Amplicons

This step uses EBLTS to tagment PCR amplicons. The tagmentation process fragments and tags the PCR 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 PP1 and PP2 were 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 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. Label a new PCR plate TAG1.
2. Combine PP1 and PP2 as follows.
a. Transfer 10 µl from each well of the PP1 plate to the corresponding well of the TAG1 plate.
b. Transfer 10 µl from each well of the PP2 plate to each well of the TAG1 plate containing starting sample from PP1.

If you are only using one primer pool, add 10 ul of nuclease-free water in place of PP2 samples to each well of the TAG1 plate containing sample from PP1 plate.

3. 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)
4. Add 30 µl Tagmentation master mix to each well in TAG1 plate.
5. Seal and shake at 1600 rpm for 1 minute.
6. Place on the preprogrammed thermal cycler and run the IMAP TAG program.