Can NotI and BamHI be digested with the same buffer? What if it's not completely cut?

In terms of brand channels, NEB's NotI-HF/BamHI-HF and Thermo's FastDigest series are both commonly used, and domestic brands like Vazyme and Novoprotein also have stock available, with relatively flexible lead times and after-sales support. If your lab only has CutSmart buffer, I would prioritize the NEB HF series; if you need it urgently, you can directly contact a local authorized distributor and have them confirm the in-stock lot numbers for NotI-HF and BamHI-HF, and request activity reports. Buy small packages for validation first, and don't purchase large packages all at once.

Double enzyme digestion can improve efficiency: first prepare the system to 100-150 μ L, add the first enzyme for 1 hour of digestion, and then add the second enzyme for another 1 hour of digestion; do not add a large number of two enzymes at once, as glycerol accumulation will affect activity. 3 μ g DNA can be added with enzymes at around 5 U/μ g, but the total enzyme volume should not exceed 10% of the system. After cutting, take 200-300 ng of rubber to confirm completeness, and then recycle it uniformly. If two sites are very close, you can linearize them first and then see if the second site is underexposed.

I have encountered incomplete NotI/BamHI digestion. The most common cause is not the buffer, but rather the NotI site being affected by CpG methylation. The NotI recognition sequence contains CpG. If this site is methylated in plasmids extracted from conventional E. coli, NotI will fail to cut or cut incompletely. I recommend first digesting the vector and insert separately with NotI and running a gel to check whether complete linearization is achieved. If the single-enzyme digest fails, adding more enzyme or extending the incubation time usually will not help. You will need to switch to a dam-/dcm- host to re-prepare the plasmid, or change the digestion strategy.

If you use NEB's NotI HF and BamHI HF, their activity in CutSmart can usually be maintained at 100% or close, and theoretically, they can be cleaved by the same buffer with dual enzymes. However, the prerequisite is to confirm that what you have in your hand is not a regular Noti/BamHI. Some regular versions may have decreased activity in CutSmart. You can first check the enzyme activity table on the official website, or make a single enzyme control: take 0.5 μ g each and cut it in CutSmart for 30 minutes to see if it can be completely linearized, and then decide whether to perform double enzyme cutting.