Amplify Library

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

This step uses PCR to amplify the anchor-ligated DNA fragments and add indexes and primer sequences for cluster generation. The resulting product is a dual-indexed library, which is a DNA fragment with adapters at each end.

For help with selecting index adapters, refer to Pooling Preparation.

• EPM (Enhanced PCR Mix)
• Index adapter plate (UDP0XXX)
• Each well of the index adapter plate is single-use and contains > 10 µl UDP0XXX, which are premixed Index 1 (i7) and Index 2 (i5) adapters.
• The row and column labels are printed on the underside of the index adapter plate. Raise the plate overhead to check the labels.
1. Prepare the following consumables:

Reagent

Storage

Instructions

EPM

-25°C to -15°C

Thaw at room temperature. Invert to mix, and then centrifuge briefly.

Index adapter plate

-25°C to -15°C

Thaw at room temperature. Vortex to mix, and then centrifuge at 1000 × g for 1 minute.
2. Save the following PCR program on a thermal cycler using the appropriate number of PCR cycles, which are listed in the following table:
• Choose the preheat lid option and set to 100°C
• Reaction volume is 50 µl
• 98°C for 30 seconds
• X cycles of:
• 98°C for 10 seconds
• 60°C for 30 seconds
• 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 4°C for ≤ 16 hours

Adjust the program to optimize for different input amount.

When multiple samples are amplified on one plate, make sure that the input for each sample is the same.

Input Amount (ng)

Number of PCR Cycles (X)

25

15

100

13

1000

10

Total program time is ~44 minutes for 15 cycles, ~39 minutes for 13 cycles, and ~33 minutes for 10 cycles.

1. If you are resuming the protocol after a safe stopping point, centrifuge the sealed PCR plate at 280 × g for 10 seconds.
2. Using a new pipette tip for each well, pierce the foil covering the index adapter plate wells that you intend to use.
3. Add the following volumes to each well of the PCR plate in the order listed. Transfer UDP0XXX from the index adapter plate to the PCR plate.
• UDP0XXX (10 µl)
• EPM (20 µl)
4. Pipette 10 times to mix, and then seal.
5. Place on the preprogrammed thermal cycler and run the PCR program.

Each well contains a volume of 50 µl.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at ‑25°C to ‑15°C for up to 7 days.