Amplify Tagmented DNA

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This step amplifies the tagmented DNA using a limited-cycle PCR program. The PCR step adds Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for sequencing cluster generation. To confirm the indexes of libraries selected for low plexity pooling have the appropriate color balance, refer to the Index Adapters Pooling Guide.

For a list of compatible index adapters for use with this protocol, refer to Product Content.

• EPM (Enhanced PCR Mix)
• 1.5 ml microcentrifuge tubes
• Index adapters
• Microseal 'B' adhesive seal
• Nuclease-free water
• Pipette tips
– 20 µl
– 200 µl
• Index adapter plates
– A well can contain > 10 µl index adapters.
– Do not add samples to the index adapter plate.
– Each well of the index plate is single use only.
1. Prepare the following consumables:
• EPM—Invert to mix, then centrifuge briefly.
• Index adapter plate:
• Use 10 base pair index codes that differ from other indexes such as Nextera DNA CD indexes, which use eight base pair index codes. Confirm that the sequencing system is configured for 10 base pair index codes.
• Centrifuge at 1000 × g for 1 minute to settle liquid away from the seal.
• Pipette slowly to minimize foaming.
• [Plates] Spin briefly before use.
• [Tubes] Vortex to mix, then centrifuge briefly.
2. Save the following BLT PCR program on a thermal cycler using the appropriate number of PCR cycles indicated in the table:
• Choose the preheat lid option and set to 100°C
• 68°C for 3 minutes
• 98°C for 3 minutes
• (X) cycles of:
• 98°C for 45 seconds
• 62°C for 30 seconds
• 68°C for 2 minutes
• 68°C for 1 minute
• Hold at 10°C

Total DNA Input (ng)

Number of PCR Cycles (X)

1–9

12

10–24

8

25–49

6

50–99

5

100–500

5

Blood/Saliva

5
Procedure
1. For each sample, combine the following volumes to prepare the PCR Master Mix. Multiply each volume by the number of samples being processed.
• EPM (22 µl)
• Nuclease-free water (22 µl)

Reagent overage is included in the volume to ensure accurate pipetting.

2. Vortex, and then centrifuge the PCR Master Mix at 280 × g for 10 seconds.
3. With the plate on the magnetic stand, use a 200 µl multichannel pipette to remove and discard supernatant.

Foam that remains on the well walls does not adversely affect the library.

4. Remove from the magnetic stand.
5. Immediately add 40 µl PCR Master Mix directly onto the beads in each sample well.
6. Immediately pipette to mix until the beads are fully resuspended. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
7. Seal the sample plate and centrifuge at 280 × g for 3 seconds.
8. Add the appropriate index adapters to each sample.

Index Kit Type

Kit Configuration

Volume of Index Adapter per Sample

96 plex (dual index)

96-well plate

10 µl prepaired i7 and i5
9. Using a pipette set to 40 µl, pipette 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
10. Seal the plate with Microseal 'B', and then centrifuge at 280 × g for 30 seconds.
11. Place on the preprogrammed thermal cycler and run the BLT PCR program.

SAFE STOPPING POINT

If you are stopping, store at 2°C to 8°C for up to 30 days.