enzymes inactivation - (May/21/2011 )

Do we have to inactivate the restriction enzymes and ligase after restriction digestion and ligation respectively, before purifying? How critical is it for successful cloning?

Sneha

If you are cloning and re-creating the cut sites (ligating an EcoRI end to another EcoRI end, e.g.) then you have to eliminate the restriction enzyme first. Heat killing is by far the easiest, and given a choice, I'd recommend choosing enzymes with that in mind. Ligation enzymes are somewhat inhibitory to transformation, but this is a relatively minor problem. You can heat kill them if you want.

Thankyou for your reply. So it basically means that we do not have to inactivate them unless situation like what you gave arises/

That's right, but I should emphasize that this is a relatively rare situation. It means that all of your ligations are ligating (say) AvrII sites to NheI sites, rather than NheI to NheI. Also, note that the vector and insert both also need to be free of all of the enzyme sites, not just the ones used to cut that specific fragment.