Protocol for use with NEBNext® Ultra™ II FS DNA Module (E7810)

This caution sign signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.
Colored bullets indicate the cap color of the reagent to be added to a reaction.
Stopping points in the protocol.

Starting Material: 100 pg–500 ng purified, genomic DNA. We recommend that the DNA be in 1X TE (10 mM Tris pH 8.0, 1 mM EDTA), however, 10 mM Tris pH 7.5–8, low EDTA TE or H2O are also acceptable. If the input DNA is less than 26 µl, add TE (provided) to a final volume of 26 µl.

1.1. Fragmentation/End Prep
Fragmentation occurs during the 37°C incubation step. Use the chart below to determine the incubation time required to generate the desired fragment sizes. Incubation time may need to be optimized for individual samples. See Figure 1.1 for a typical fragmentation pattern.

 100 bp-250 bp
 30 min
 30-40 min
 150 bp-350 bp
 20 min
 20-30 min
 200 bp-450 bp
 15 min
 15-20 min
 300 bp-700 bp
 10 min
 5-15 min
 500 bp-1 kb  5 min  5-10 min

Figure 1.1: Example of size distribution on a Bioanalyzer®. Human DNA (NA19240) was fragmented for 5-40 min.

1.1.1. Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.

1.1.2. Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice.

Note: It is important to vortex the enzyme mix prior to use for optimal performance.

1.1.3. Add the following components to a 0.2 ml thin wall PCR tube on ice:

 DNA  26 µl
(yellow) NEBNext Ultra II FS Reaction Buffer
 7 µl
(yellow) NEBNext Ultra II FS Enzyme Mix
 2 µl
 Total Volume
 35 µl
1.1.4. Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.
1.1.5. In a Thermocycler, with the heated lid set to 75°C, run the following program:

5–30 min @ 37°C
30 min @ 65°C
Hold @ 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 using the NEBNext Ultra II Ligation Module (NEB #E7595) before stopping.