XbaI cannot cleave methylated plasmids. Is there a recommended alternative restriction enzyme?

Alternative enzymes that also generate CTAG sticky ends include SpeI, NheI, or AvrII. These enzymes are not as sensitive to dam methylation as XbaI, and their sticky ends are compatible, so they can be used in the original construction scheme. However, before substitution, be sure to check the vector and insert sequences for additional internal sites of the new enzyme; otherwise, the DNA may be cut into small fragments. Digestion efficiency is usually not significantly lower than that of XbaI, but because different site sequences vary, it is recommended to perform a time-course validation.

If XbaI cannot be cut, do not just change the enzyme. It is recommended to first consider changing the dam -/dcm host to transform and extract the plasmid. Because XbaI is usually affected by dam methylation near the site sequence, after switching hosts, the original XbaI site can often resume cleavage, and the subsequent connecting sequence will not change. This method is more direct and cost-effective than enzyme exchange. If you have the dam -/dcm receptive state on hand, I will prioritize doing this instead of changing the primer or transposition point.

Within the budget range you mentioned, you can look for NheI/SpeI small packaging from NEB, Thermo, or Takara, and domestic nearshore and Norwegian brands are also commonly available in stock. When contacting the agent, explain that it is for regular cloning and request confirmation of availability and batch inspection reports. Personal experience shows that enzyme substitution efficiency is not low, but do not buy large packages all at once; Buy the smallest packaging first, take 1 μ g plasmid for 1-hour enzyme digestion test, run the gel to confirm complete linearization.

Note that even with compatible cohesive ends, after ligation of XbaI with SpeI/NheI, the original site may no longer be recognized by the original enzyme; for subsequent identification, switch to sequencing or an alternative primer strategy. It is recommended to set up controls: digest the same batch of plasmid separately with XbaI and the new enzyme, and compare by gel electrophoresis; after ligation, transform, pick several clones for colony PCR and sequencing confirmation. This will verify whether the substitute enzyme is suitable for your construction, so you won't discover a ligation error only at the final sequencing step.