Can domestic restriction endonucleases be used for double digestion as a substitute for NEB's?

Regarding star activity and buffer compatibility, I suggest not doing a large-scale replacement right away. First, take the same batch of plasmid substrate and perform EcoRI/HindIII double digestion with domestic enzymes, setting the incubation time at 30 min, 1 h, and 3 h, then run electrophoresis to check for extra bands or smearing. EcoRI is sensitive to glycerol and ionic conditions, and star activity tends to be more pronounced in some domestic universal buffers; you can ask the supplier to provide the enzyme activity definition and star activity test conditions. For double digestion buffer compatibility, verify that both enzymes retain more than 80% activity, otherwise incomplete digestion is likely. Our lab has used domestic enzymes; plasmid linearization was fine, but for PCR product digestion, you must leave 6 protective bases at both ends. With domestic enzymes, the drop in efficiency is more obvious when terminal protective bases are insufficient. I suggest requesting a trial sample first rather than buying a large package all at once.

From a supply and cost perspective, there are quite a few domestic EcoRI/HindIII options within a budget of 300 yuan per unit. It is advisable to prioritize checking whether there is stock and a batch-specific COA. With a usage of twenty to thirty times per week, stable lead times are more important than unit price. Ask a local distributor clearly: whether the same batch can be supplied continuously, whether trial samples can be provided, and what the return and exchange policy is. When we switched to domestic products, we first had three companies each send small packages, tested double digestion on the same batch of plasmid, and then used the recovered fragments for ligation. Ligation efficiency can reflect end integrity. In terms of brands, Nearshore, Vazyme, and TransGen can all be inquired about, but which one is actually stable should be determined based on validation with the plasmid you have on hand. If it is only for routine cloning, domestic products can usually cover the need; if you are doing library construction or sequencing, it is advisable to keep NEB for critical batches.

Regarding the efficiency of cutting PCR products, I suggest not only running the gel after restriction digestion, but also performing a side-by-side comparison with NEB. In our experience, domestic EcoRI/HindIII can often handle double digestion of plasmids, but cutting PCR products is more prone to incomplete digestion or nonspecific cleavage, because the terminal state of PCR products and the design of protective bases have a major impact. It is recommended to include at least 6 protective bases at both ends of the primers, avoid excessive enzyme addition, take a sample for electrophoresis after 1 h digestion at 37°C, and then add more enzyme. Do not mix domestic 10×Buffer across brands, especially for double digestion; using the matching buffer from the same brand is more reliable. If smearing occurs, first reduce the final glycerol concentration (enzyme volume should not exceed 1/10) and shorten the time, which usually improves it. Overall, for routine high-volume use, it can be substituted, but keep one vial of NEB as a control for troubleshooting abnormalities.