Prepare cfDNA and Perform End Repair

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This step converts the 5' and 3' overhangs on cfDNA into blunt ends using an end repair mix (ERP6). The exonuclease activity of ERP6 removes the 3' overhangs, and the 5' to 3' polymerase activity fills in the 5' overhangs. A phosphate is added to the 5' end, in preparation for A-Tailing.

• ERP6 (End Repair Mix 6)
• RSB (Resuspension Buffer)
• Microseal 'B' adhesive seal
• 96-well PCR plate, semiskirted
1. Prepare the cfDNA samples:
a. Vortex to mix, and then centrifuge briefly.
b. Use RSB to prepare 20 ng of each purified cfDNA sample in a final volume of 45 μl (0.44 ng/μl).

Refer to cfDNA Input Recommendations for more information.

c. Pipette or vortex to mix, and then centrifuge briefly.
2. Prepare the following consumables:
• ERP6—Vortex to mix, and then centrifuge briefly.
• RSB—Vortex to mix.
3. Label a new 96-well semiskirted PCR plate LP.
4. Save the following ERP program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 55 µl
• 30°C for 30 minutes
• 70°C for 10 minutes
• 4°C for 1 minute
• Hold at 4°C
1. Transfer 45 μl from each prepared cfDNA sample to the corresponding well of the LP plate.
2. Add 10 μl ERP6 to each well of the LP plate, and pipette 10 times to mix.
3. Apply Microseal 'B', and then centrifuge briefly.
4. Place on the preprogrammed thermal cycler and run the ERP program.
5. When the program reaches the 4°C hold, immediately proceed to the next step.