restriction enzyme inactivation - How do you inactivate restriction enzymes? (Jul/05/2005 )

Hi!

I wonder how you best inactivate restriction enzymes after digestion? Recently, in our lab we put the tubes in the freezer over night, and then an ethanol precipition follows.

Is there any other (faster?) way? Or is it unecessary, do the enzymes become inactivated during the ethanol step anyway?

Best way to inactive an enzyme is to heat the sample. Usually 65C for 10min does the job, but it best to look up the recommended time and temp for each enzyme. Promega (I am pretty sure) has a section on this.

I have never tested activation of an enzyme in ethanol so I can tell you that. I would guess that it would be and when you ppt your plasmid in the ethanol, the enzyme will stay in the supernate.

You can use 1 uL of 0,5 M EDTA and 65C for 10min.

NEB (New England Biolabs) catalog tells how to inactivate each of their enzymes. This info is available on line as well.

I use the PCR purification kit from Qiagen, which is very fast