Tagment Genomic DNA

This snippet causes dropdowns to open on page load. This text/paragraph does not appear in the output.

This step uses the Bead-Linked Transposomes, Long Range (BLT-LR) to tagment DNA. This process fragments and tags the DNA with adapter sequences.

• Bead-Linked Transposomes, Long Range (BLT-LR)
• Resuspension Buffer (RSB)
• Tagmentation Buffer 1 (TB1)
• 1.7 ml microcentrifuge tube
• 96-well PCR plate

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.

• BLT-LR remains in wells until after purification.
• BLT-LR must be stored at temperatures above 2°C. Do not use BLT-LR that has been stored below 2°C.
1. Prepare the following consumables:
• BLT-LR—Vortex to mix. Do not centrifuge before pipetting.
• RSB—Vortex to mix.
• TB1—Vortex to mix, and then centrifuge briefly.
2. Save the following TAG program on the thermal cycler:
• Choose the preheat lid option and set to 80°C
• Set the reaction volume to 50 µl
• 55°C for 5 minutes
• Hold at 10°C
3. Label a new 96-well PCR plate TAG.
1. Add DNA to each well of the TAG plate as follows.
a. Add 1-30 µl DNA to each well, so that the total input amount is within the desired range (10–1000 ng).

The recommended gDNA input is 50 ng.

b. If sample volume is < 30 µl, bring the total volume to 30 µl using RSB.
2. Vortex BLT-LR to resuspend.
3. In a 1.7 ml tube, combine the following volumes to prepare Tagmentation Master Mix. Multiply each volume by the number of samples.
• TB1 (11 µl)
• BLT-LR (11 µl)

These volumes produce 22 µl Tagmentation Master Mix per sample, which includes extra volume for accurate pipetting.

4. Pipette or vortex to mix.
5. Add 20 μl Tagmentation Master Mix to each well of the TAG plate.
6. Pipette to mix.
7. Place on the preprogrammed thermal cycler and run the TAG program.

BLT-LR remain in wells until after purification.