NEBNext Ultra II FS DNA Module E7810
Juliet Bonnevie
Abstract
The NEBNext Ultra II FS DNA Module contains the enzymes and buffers required to convert a broad range of input amounts of intact DNA into fragmented DNA with 5´ phosphorylated 3´ dA-tailed ends. The module is optimized for use with the NEBNext Ultra II Ligation Module (NEB #E7595) and with the NEBNext Ultra II Q5 Master Mix (NEB #M0544) if amplification is required. The fast, user-friendly workflow has minimal hands on time.
Note: The Ultra II FS Module is not compatible with bisulfite conversion workflows
Each module component must pass rigorous control standards, and for each new lot the entire set of reagents is functionally validated together with NEB #E7595 and NEB #M0544 to construct indexed libraries that are sequenced on an Illumina sequencing platform.
For larger volume requirements, customized and bulk packaging is available by purchasing through OEM & Custom Solutions at NEB. Please contact custom@neb.com for further information.
Before start
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.
Attachments
Steps
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 for a typical fragmentation pattern.
| A | B | C |
|---|---|---|
| Fragmentation Size | Incubation @ 37°C | Optimization |
| 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: Example of size distribution on a Bioanalyzer®. Human DNA (NA19240) was fragmented for 5–40 mins.
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.
Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice.
Add the following components to a 0.2 ml thin wall PCR tube on ice:
| A | B |
|---|---|
| Component | Volume per One Library |
| DNA | 26 µl |
| NEBNext Ultra II FS Reaction Buffer | 7 µl |
| NEBNext Ultra II FS Enzyme Mix | 2 µl |
| Total Volume | 35 µl |
Vortex the reaction for 0h 0m 5s and briefly spin in a microcentrifuge.
In a thermocylcer, with the heated lid set to 75°C, run the following program:
5-30min @ 37°C
@ 0h 30m 0s @ 65°C
Hold @ 4°C
