I have been trying to use NEB's TtAgo (https://www.neb.com/products/m0665-ttago) to linearize supercoiled plasmids, following NEB's recommendations, but I haven't had any success yet (19/19 failed reactions). I am in touch with NEB Technical Support, they have answered most of my questions, but they haven't been able to provide me with a full working protocol, which would be very helpful to understand what I'm doing wrong.

I am using pUC19 and DNA guides that in complex with TtAgo ressemble BamHI activity. In the attached file, lane M1 is a 1kb DNA ladder, lane M2 is BsaI-linearized pUC19, lane 1 is undigested plasmid, lane 2 a temperature control, lane 3 is plasmid treated with apo-TtAgo, and lane 4 is plasmid treated with TtAgo-gDNA complexes. If the experiment worked, at least lane 4 should look like lane M2.

Has anynone successfully used TtAgo for any type of application? If so, what are the main parameters you need to control in order to get your experiment to work (incubation time, incubation temperature, buffer system, ratios, etc.)?