Digestion with NEBNext dsDNA Fragmentase (M0348)
New England Biolabs
Abstract
NEBNext dsDNA Fragmentase is an enzyme-based reagent that shears DNA to produce fragments of the desired sizes in a time-dependent manner, for next generation sequencing library preparation protocols
- dsDNA Fragmentase provides random fragmentation, similar to mechanical methods (1,2).
Before start
Adequate mixing of NEBNext dsDNA Fragmentase is important for the success of this reaction. NEBNext dsDNA Fragmentase should be vortexed for 0h 0m 3s prior to use .
For tough digestions, add 1µL of 200millimolar (mM) to the reaction. Additional MgCl2 can be added if necessary.
The protocol listed below is for fragmentation of 5 ng–3 μg of DNA.
Steps
Vortex NEBNext dsDNA Fragmentase for 0h 0m 3s, quick spin and place 37On ice.
Combine the following components in a sterile PCR tube and vortex:
| A | B |
|---|---|
| Component | Amount |
| DNA (5 ng–3 μg) | 1–16 μl |
| 10X Fragmentase Reaction Buffer v2 | 2 μl |
| Sterile Water | variable |
| Final Volume | 18 μl |
Add 2µL and vortex mixture for 0h 0m 3s.
Incubate at 37°C for the recommended times below to generate the desired fragment size:
| A | B |
|---|---|
| Desired Fragment Size (bp) | Incubation Time (min) |
| 50–200 | 25–35 |
| 200–1,000 | 15–25 |
| 1,000–2,000 | 10–15 |
Add 5µL to stop the reaction.
Clean up the fragmented DNA with column purification or using SPRI beads.
For further analysis:
Bioanalyzer: Clean up the fragmented DNA prior to loading on a Bioanalyzer chip.
End Repair : Clean up the fragmented DNA then proceed with desired DNA end repair protocol.
Polyacrylamide Gel Analysis: Clean up the fragmented DNA prior to loading the samples on a PAGE gel.
Long Term Storage: Clean up the fragmented DNA prior to long term storage.
Agarose Gel Size Selection/Analysis : Samples can be loaded directly on to an agarose gel. It is not necessary to clean up the reactions prior to loading.
