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Digestion with NEBNext dsDNA Fragmentase (M0348) |  | | Digestion with NEBNext dsDNA Fragmentase (M0348) |
Introduction
Careful preparation and concentration determination of the starting material are important for the success of this reaction.
The recommended range for starting material is 1-5 µg but lower or higher amounts can be used. The final DNA concentration in the reaction should be 0.05 µg/µl. Incubate reaction mix on ice for 5 minutes before adding NEBNext dsDNA Fragmentase. Add 2 µl of NEBNext dsDNA Fragmentase per µg DNA and incubate as described below.
Protocol
- Vortex DNA sample.
- Set up the digestion reaction using the following guidelines (note that NEBNext dsDNA Fragmentase should not be added at this point):
| Reaction Components | Starting DNA Amount (µg) | | DNA (µg) | 1 | 2 | 3 | 4 | 5 | | 10X Fragmentase Reaction Buffer (µl) | 2 | 4 | 6 | 8 | 10 | | 100X BSA (µl) | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 | | dsDNA Fragmentase (µl) | 2 | 4 | 6 | 8 | 10 | | Final Volume* (µl) | 20 | 40 | 60 | 80 | 100 | - Vortex thoroughly.
- Incubate on ice for 5 minutes.
- Vortex NEBNext dsDNA Fragmentase and add enzyme to the reaction.
- Vortex thoroughly.
- Incubate at 37°C according to the recommended times below to generate the desired fragment size (see notes).
| Desired Fragment Size (bp) | Incubation Time (min) | | 600-800 | 15 | | 300-600 | 20 | | 100-300 | 30 | - Add 5 µl of 0.5 M EDTA to stop the reaction.
- DNA fragments are ready for DNA end repair, size selection, or analysis.
End Repair: Clean up the fragmented DNA (e.g. column purification) then proceed with desired DNA end repair protocol. If DNA fragments are to be used for the Illumina® Genome Analyzer DNA sample preparation, add 1 µl of E. coli DNA Ligase for Fragmentase to the end repair reaction with ≥ 1 µg DNA or 0.5 µl of E. coli DNA Ligase for Fragmentase to the end repair reaction with < 1 µg DNA.
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 purification) prior to long term storage.
Notes: For gDNA containing > 60% GC content, a longer incubation time with dsDNA Fragmentase is required to obtain the desired fragment size.
PCR products and cDNA require a different incubation time with dsDNA Fragmentase to obtain the desired fragment size.
The E. coli DNA Ligase for Fragmentase contains NAD and is provided only for use in DNA end repair reactions following NEBNext dsDNA Fragmentase incubation and DNA clean up. Do not add E. coli DNA Ligase to the NEBNext dsDNA Fragmentase reaction.
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