Hi there,

Phenol extraction and ethanol precipitation is the alternative for heat resistant activities.

Since most restriction enzyme activity is dependent on the presence of divalent metal ions(magnesium), using EDTA could also inactive them.

I am not sure if this is a proper answer. If the ends are blunt maybe there will be no restriction. The Mg+2 is a cofactor and EDTA are chelators, they restrict the contents,like Mg+2. To bare in mind the STAR activity.(maybe it helps if you are just attending one enzyme). I think they are some more but I don't remember them now. Are you not by heat? And maybe HSP(60 and 70).

Zahra Sabouri

Dear Zahra Sabouri

There inst necessity to inactivate restriction enzyme if you load your digestion on agarose gel and then purify desired band from gel. By loading digest reaction on agarose gel simply enzyme remove from reaction and you could move forward with ligation without any problem. I recommend to just try gel purification of digestion products.

Any way, there are also some other ways which could be used which are not really necessary. Based on NEB, using a column for cleanup (such as the Monarch® PCR & DNA Cleanup Kit), or performing a phenol/chloroform extraction could be effective in the case of sensitive enzyme to heating.

Base on TFS and TAKARA sites sfiI enzyme could be inactivated easily by heat inactivation on 70 C.

Good luck

(unless you are looking for a different way if inactivation and not by heating. Thermolabile or sensitivity to heat is just one step. Could you make by metabolic engineering an inhibitor?)