Can the rapid endonuclease cleave overnight and will star activity occur?

I have prepared it using rapid enzymes here, but it is generally not recommended to stay overnight. Fast enzyme activity is strong, glycerol concentration is high, and long-term reaction is prone to star formation, especially in the case of low DNA content. When the enzyme amount is high, the background will be obvious when the enzyme amount is high in the system of 50-100 μ L. It is recommended to follow the instructions for 15 minutes, with a maximum extension of 1 hour. If you want to cut multiple tubes at once, you can divide them into several tubes in the same system, quickly cut each tube, merge them, and then recycle them uniformly. This is more efficient and less prone to non-specific cutting.

Before using a fast enzyme overnight, first confirm whether the buffer is suitable for long reactions. Some fast enzymes have limited stability at 37°C in the recommended buffer, and may partially lose activity after overnight incubation, resulting in incomplete digestion rather than star activity. It is advisable to set up a control: the same DNA digested overnight with a regular enzyme vs. 15 min with a fast enzyme, and compare completeness. For 2 μg of plasmid, a single fast enzyme digestion is usually sufficient for subsequent gel extraction, and overnight digestion is unnecessary. If you only have a fast enzyme on hand, you can also perform multiple rounds of fast digestion and then pool the products, which is more controllable than risking an overnight reaction.

I have encountered star activity myself, mainly when digesting overnight and when the enzyme amount is too high. The buffer for fast enzymes is not necessarily fully universal. The Thermo FastDigest series uses a universal buffer, while the NEB HF series mostly uses CutSmart, but you need to check the instructions for different product lines. Before switching brands, it is best to run a small test first: take 0.5 μg of plasmid, digest for 30 min, 2 h, and overnight respectively, and run a gel to see whether there is smearing or extra bands. If the overnight lane has a clean background, then scale up to 2 μg for preparation; if there are extra bands, do not digest overnight.

If the star band has already been cut overnight, it is usually not recommended to remedy it again, as star cutting may generate random incisions inside the target fragment, and the connection is prone to missing or mutating after retrieval. It is more reliable to perform enzyme digestion again or to cut the gel to recover the target band, but it needs to be confirmed that the target band has not been further cut. I still prefer to use regular enzymes overnight for construction, while fast enzymes are only used for identification or short-term preparation. If it is necessary to stay overnight, the enzyme dosage can be reduced to the lower limit recommended in the instructions, and the proportion of glycerol volume can be reduced.