Manufacturer’s instructions

2.1 In the same plate/tube(s) in which enzymatic fragmentation was performed, assemble each end repair and A-tailing reaction as follows:

Component Volume 1/2 rxn
Fragmented, double-stranded DNA 50 μL 25 μL
End Repair & A-Tailing Buffer* 7 μL 3.5 μL
End Repair & A-Tailing Enzyme Mix* 3 μL 1.5 μL
Total volume: 60 μL 30 μL

The buffer and enzyme mix should preferably be pre-mixed and added in a single pipetting step. Premixes are stable for ≤24 hrs at room temperature, for ≤3 days at 4°C, and for ≤4 weeks at -20°C.

2.2 Vortex gently and spin down briefly. Return the reaction plate/tube(s) to ice. Proceed immediately to the next step.

2.3 Incubate in a thermocycler programmed as outlined below. A heated lid is required for this step. If possible, set the temperature of the heated lid to ~85°C (instead of the usual 105°C).

Step Temp Time
End repair and A-tailing 2 65 °C 30 min
Hold 4°C ∞

- Both the fragmentation and end repair enzymes are inactivated at 65°C. When reactions are set up according to recommendations, additional fragmentation should be negligible. The brief period of end repair is sufficient for enzymatically fragmented DNA.

- If proceeding to the adapter ligation reaction setup without any delay, the reaction may be cooled to 20°C instead of 4°C.

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