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.
End Repair: Clean up the fragmented DNA (e.g.
column purification, or using SPRI) then proceed
with desired DNA end repair protocol.
- 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.
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.