Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community.
Tip: Adequate mixing of NEBNext dsDNA Fragmentase is important for the success of this reaction. NEBNext dsDNA Fragmentase should be vortexed for 3 seconds prior to use.
For tough digestions, add 1 μl of 200 mM MgCl2 to the reaction. Additional MgCl2 can be added if necessary.
The protocol listed below is for fragmentation of 5 ng–3 μg of DNA.
- Vortex NEBNext dsDNA Fragmentase for 3 seconds, quick spin and place on ice.
- Mix together the following components in a sterile PCR tube:
|DNA (5 ng–3 μg)
|10X Fragmentase Reaction Buffer v2
- Add 2.0 μl dsDNA Fragmentase and vortex the mixture for 3 seconds.
- Incubate at 37°C for the recommended times below to generate the desired fragment size.
*If starting material is 100 ng or less, incubation times should be increased by 10 minutes.
|Desired Fragment Size (bp)
||Incubation Time (min)
- Add 5 μl of 0.5 M EDTA to stop the reaction.
- DNA fragments are ready for DNA end repair, size selection or analysis.
Clean up the fragmented DNA (e.g.
column purification, or using SPRI) then proceed
with desired DNA end repair protocol.
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.
Polyacrylamide Gel Analysis:
Clean up the fragmented DNA (e.g. column purification) prior to loading the samples on a PAGE gel.
Long Term Storage:
Clean up the fragmented DNA (e.g. column purifications, or SPRI Beads*) prior to long term storage.
*Note: If using SPRI Beads for sample purification, it is recommended to dilute the sample 1:1 with sterile water to allow for faster collection of beads to the magnet.