Amplify Libraries
This step amplifies the tagmented DNA using a limited-cycle PCR program. The PCR step adds the Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for sequencing cluster generation. To confirm indexes selected for low plexity pooling have the appropriate color balance, refer to the Index Adapters Pooling Guide.
Index adapter tubes or plates are ordered separately from the library prep components. For a list of compatible index adapters for use with this protocol, refer to Illumina-Supplied Consumables.
Consumables
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NPM (Nextera PCR Master Mix) |
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Index adapters (tubes or plates) |
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Microseal 'A' adhesive film |
About Reagents
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A well may contain > 10 µl index adapters. |
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Do not add samples to the index adapter plate. |
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Each well of the index plate is single use only. |
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Open only one index adapter tube at a time to prevent misplacing caps. Alternatively, use fresh caps after opening each tube. |
Preparation
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1.
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Prepare the following consumables: |
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Index adapters
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‑25°C to ‑15°C
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Thaw at room temperature.
[Tubes] Vortex to mix, and then centrifuge briefly.
[Plates] Spin briefly before use.
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NPM
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‑25°C to ‑15°C
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Thaw on ice for 20 minutes.
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2.
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Save the following NXT PCR program on a thermal cycler: |
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Choose the preheat lid option and set to 100°C |
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Set the reaction volume to 50 µl |
Procedure
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1.
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Add the following index adapter volumes per sample according to your index adapter kit type. |
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Index adapter tubes
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5 µl i7 adapter
5 µl i5 adapter
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Index adapter plate
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10 µl prepared i7 and i5 index adapters
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2.
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Add 15 µl NPM to each well. |
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3.
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Pipette 10 times to mix, and then seal the plate. |
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4.
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Centrifuge at 280 × g at 20°C for 1 minute. |
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5.
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Place on the preprogrammed thermal cycler and run the NXT PCR program. |
SAFE STOPPING POINT
If you are stopping, seal the plate, and store at 2°C to 8°C for up to 2 days. Alternatively, leave on the thermal cycler overnight.
