What can cause over fragmentation of DNA samples?
Improper fragmentation time is one cause of over fragmentation; see the CoA or the product box for the recommended lot-specific fragmentation time for supported fragment sizes.
Over digestion is another cause and can occur if the reaction is left at room temperature.
Make sure the Enzymatic Prep Master Mix and the DNA sample are kept on ice until placed onto the pre-chilled thermocycler. Over digestion can occur when sample integrity is compromised.
Fragmentation time must be adjusted for DNA samples that are not high molecular weight (e.g., FFPE). We have observed more rapid digestion for FFPE samples.
Over digestion can also occur if your DNA sample storage buffer contains no EDTA, such as using a 10 mM Tris buffer. Fragmentation times are determined using a low EDTA TE storage buffer with 0.1 mM EDTA. In this case, do not add Reagent K2 to your fragmentation reaction, and additional optimization may be required.