If the goal is to obtain DNA of maximal size, shaking during lysis in the thermal mixer is not required. However, Proteinase K digestion needs to be carried out at 56°C. To ensure proper lysis, invert your sample a number of times slowly during the proteinase K incubation. Please note that samples lysed without agitation will be very viscous and difficult to handle. If the goal is DNA tuned to 50 kb-250 kb (optimal for ligation-based nanopore sequencing), then we strongly recommend working with a thermal mixer (e.g., Eppendorf® ThermoMixer® C). This will result in a controlled shearing to the optimal fragment size range. If access to a thermal mixer is not possible, the best alternative for agitation in the thermal mixer is to pulse-vortex for one minute at the highest speed possible after the lysis incubation. For tissue samples, briefly vortex the sample every minute or so during the beginning of the lysis incubation, until there are no tissue pieces remaining. Then, vortex again for one minute after the lysis incubation.