Amplify Tagmented DNA

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

This step amplifies the tagmented DNA using a limited-cycle PCR program. The PCR step adds prepaired 10 base pair Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for library clustering on the flow cells. To confirm the indexes of libraries being pooled for enrichment 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 Consumables & Equipment.

• EPM (Enhanced PCR Mix)
• Index adapter plate
• Nuclease-free water
• Low DNA binding PCR plate
• 1.7 ml microcentrifuge tubes
• Microseal 'B' adhesive seal
• Pipette tips
– 20 µl multichannel pipettes
– 200 µl multichannel pipettes
• Index adapter plates
– A well may contain > 10 µl index adapters.
– Do not add samples to the index adapter plate.
– Each well of the index plate is single use only. Refer to Tips and Techniques for best practices when working with index plates.
– You can use plates for a maximum of four freeze-thaw cycles if not using all 96 indexes in a single experiment.
– Each index plate well contains a unique, prepared mix of Index 1 (i7) and Index 2 (i5).
1. Prepare the following consumables:

Item

Storage

Instructions

EPM

‑25°C to ‑15°C

Thaw on ice. Invert to mix, then centrifuge briefly.

Index adapter plate

‑25°C to ‑15°C

Thaw at room temperature, then keep on ice.
2. Save the following eBLT 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
• Set the reaction volume to 50 µl
• 72°C for 3 minutes
• 98°C for 3 minutes

Note the initial 72°C incubation before the 98°C denaturation step. Insufficient incubation can result in unsuccessful amplification.

• (X) cycles of:
• 98°C for 20 seconds
• 60°C for 30 seconds
• 72°C for 1 minute
• 72°C for 3 minutes
• Hold at 10°C

The total running time is ~38 minutes for 9 cycles and ~46 minutes for 12 cycles.

Sample Input Type

Number of PCR Cycles (X)

10–49 ng genomic DNA

12

50–1000 ng genomic DNA

9

Saliva

9

Blood

9
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 (23 µl)
• Nuclease-free water (23 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 set to 100 µl 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 10 times 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 10 seconds.
8. Centrifuge the index adapter plate at 1000 × g for 1 minute.
9. Prepare the index adapter plate.
• [ < 96 samples] Pierce the foil seal on the index adapter plate with a new pipette tip for each well. Pierce only the number of samples being processed.
• [96 samples] Align a new Low DNA binding PCR Plate above the index adapter plate and press down to puncture the foil seal. Discard the Eppendorf PCR plate used to puncture the foil seal.
10. Using a new pipette tip, add 10 µl prepaired Index 1 (i7) and Index 2 (i5) index adapters to each well.
11. Using a pipette set to 40 µl, pipette 10 times to mix, and then seal the plate with Microseal 'B'.

Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute. Make sure that the beads are fully resuspended before proceeding to the next step.

12. Centrifuge at 280 × g for 30 seconds.
13. Place on the preprogrammed thermal cycler and run the eBLT PCR program.

SAFE STOPPING POINT

If you are stopping, store at -25°C to -15°C for up to 30 days.