Is there a significant difference in cutting efficiency between domestically produced restriction endonucleases and imported ones? Due to limited budget, I want to switch to domestic products

Perform conventional EcoRI cutting of 1-2 μ g plasmids. The gap between domestic mainstream brand conventional enzymes and imported ones is not as big as before, but inter batch differences should be noted. Suggest purchasing small packages and conducting parallel control: same batch of plasmids, same buffer, same enzyme amount, cut NEB and domestic for 30-60 minutes each, run the gel to see if the bands are complete and if there is background dispersion. If the results are consistent, using domestically produced enzymes for routine cloning is usually acceptable, but it is best to revalidate each new batch.

For the validation method, I suggest performing a time gradient and ligation test. For the time gradient, use 5, 15, 30, and 60 min to see whether the domestic enzyme tends to develop star activity or nonspecific bands after prolonged incubation. The ligation test is more critical: ligate and transform the digested vector and insert, then compare the number of positive clones and the empty vector background. Only if the ligation efficiency is close to that of the imported enzyme can it truly be considered replaceable; looking only at electrophoresis is far from sufficient.

For routine cloning, domestic enzymes can be used first, but for critical constructs, complex templates, or when high-fidelity ends are required, I still keep a tube of imported enzyme for verification or final construction. EcoRI star activity is often associated with excessive enzyme amount, high glycerol proportion, or low-salt conditions. For domestic enzymes, it is even more necessary to control the enzyme volume to no more than 10% of the system, and do not greatly extend the reaction time just to achieve complete digestion. If nonspecific bands appear, first reduce the enzyme amount or shorten the time, rather than directly switching enzymes.

When choosing domestic enzymes, I will focus on three dimensions: whether the batch inspection report is public, whether a universal buffer is provided, and the after-sales response speed of the agent. There are commonly used restriction enzyme product lines in nearshore, Novozymes, Full Gold, etc. You can buy small packages and try them out one round each. Please clarify the definition of activity, storage conditions, and delivery time, especially whether local agents can provide next day delivery. Don't just focus on cheap prices, unstable services can make experiments more troublesome.