Amir Jalilvand-Nejad I don't know what brand name is your restriction enzymes. If from New England Lab, then from its double-digestion checker, you should use NEBuffer 2.1 for double digestion. If they don't have comparable buffer, then you should use sequential digestion method.

Yuan-Yeu Yau I agree with Yuan. YOu can check the manufacturer's manual and optimum conditions from various open-source tools. Please specify your final working volume, as buffer volume depends on that and it must be 1X. The amount of enzyme depends on how much DNA you want to digest. Check for the star activity also due to which enzyme efficiency reduces.

I am sharing a link, I hope it will be useful to you.

https://www.thermofisher.com/in/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/7-common-issues-with-restriction-digestion-reactions-and-how-to-avoid-them.html

Hi Amir Jalilvand-Nejad , after you have checked that your solutions are in order like suggested by Yuan and Simant, I would definitely start by increasing the incubation time to at least 3hours - this is what we usually do in the lab.

If you have access, personally I like to use the pcr machine or heat-blocks for incubation rather than the incubator, because I feel that they are more precise and since they are directly in contact with the tubes, the samples are brought to temperature quicker.

Good luck!