Protocol for use with NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)
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Starting Material: 500 pg–1 μg fragmented DNA. We recommend that DNA be sheared in 1X TE. If the DNA volume post shearing is less than 50 μl, add 1X TE to a final volume of 50 μl. Alternatively, 10 mM Tris-HCl, pH 8.0 or 0.1X TE can be used.
1.1 NEBNext End Prep
- Mix the following components in a sterile nuclease-free tube:
(green) NEBNext Ultra II End Prep Enzyme Mix 3 μl
(green) NEBNext Ultra II End Prep Reaction Buffer 7μl
Fragmented DNA 50 μl
Total volume 60 μl
- Set a 100 μl or 200 μl pipette to 50 μl and then gently pipette the entire volume up and down at least 10 times to mix throughly. Perform a quick spin to collect all liquid from the sides of the tube.
Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
- Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program:
30 minutes @ 20°C
30 minutes @ 65°C
Hold at 4°C
If necessary, samples can be stored at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.
- Proceed directly to NEBNext Ultra II Ligation Module NEB #E7595.