TITLE: NEBNext Ultra II FS DNA Module E7810
AUTHORS: jbonnevie

[DESCRIPTION]
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.

[GUIDELINES]
Safe Stop: This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol. 

Caution: Signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.

Color: A color listed before or after a reagent name indicates the cap color of the reagent to be added.

The NEBNext Ultra II FS DNA Module is Designed for use with the Following: 
NEBNext Ultra II Ligation Module (NEB #E7595) 
NEBNext Ultra II Q5® Master Mix (NEB #M0544) 
NEBNext Singleplex or Multiplex Oligos for Illumina® 
     NEBNext Oligo Kit options can be found athttp://www.neb.com/oligos.
    Alternatively, customer supplied adaptor and primers can be used; please see www.neb.com/faq-nonNEB-adaptos.  

[STEPS]
SECTION: Fragmentation/End Prep
1. 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.
 
   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.
a




SECTION: Fragmentation/End Prep
2. 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.
 
SECTION: Fragmentation/End Prep
3. Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice.
 
 


SECTION: Fragmentation/End Prep
4. Add the following components to a 0.2 ml thin wall PCR tube on ice: 
   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   
 
 
SECTION: Fragmentation/End Prep
5. Vortex the reaction for 5 s and briefly spin in a microcentrifuge.

SECTION: Fragmentation/End Prep
6. In a thermocylcer, with the heated lid set to 75 °C, run the following program:
 
5-30min @ 37 °C
30 min @ 65 °C
Hold @ 4 °C