Can enzyme digestion and ligation be completed in one step? When will it be separated and completed in one step?

Not under any circumstances. Because restriction enzymes need to be inactivated first, otherwise the efficiency is too low.

1. If it is single enzyme digestion, dephosphorylation is needed, and some can be done together with enzyme digestion. However, dephosphorylation enzyme needs to be inactivated before it can be used for ligation, so it cannot be completed in one step.

2. If you do double enzyme digestion, or cut many bases, you must do glue recycling, otherwise once ligase enters, the cut fragments will be taken back.

Don't be afraid of trouble, the connection is still an important step. The recovery process after enzyme digestion can be slightly simplified. If only a few bases are simply cut and cannot be separated by electrophoresis, simply use PCR cleaning kit to purify and remove the enzyme. Otherwise, be honest and take it step by step.