Perform A-Tailing

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This step adds a single 'A' nucleotide to the 3' ends of blunt fragments. The A-tail prevents the fragments from ligating to each other during the adapter ligation reaction. A corresponding single 'T' nucleotide on the 3' end of the adapter provides a complementary overhang for ligating the adapter to the fragment.

• ATL4 (A-Tailing Mix 4)
• Microseal 'B' adhesive seals
1. Prepare ATL4—vortex briefly on medium-high speed to mix, and then centrifuge briefly.
2. Save the following ATL program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 90 μl
• 37°C for 20 minutes
• 70°C for 5 minutes
• 4°C for 1 minute
• Hold at 4°C
1. Add 35 μl ATL4 to each well of the LP plate, and pipette 10 times to mix.
2. Apply Microseal 'B', and then centrifuge briefly.
3. Place on the preprogrammed thermal cycler and run the ATL program.
4. When the program reaches the 4°C hold, immediately proceed to the next step.