Hi

this link can help you

https://www.neb.com/tools-and-resources/usage-guidelines/tips-for-maximizing-ligation-efficiencies

Thanjs Mangal.

Do you have any protocol related? Is this process going to removed both enzymes?

Thanks

Thanks alot Mangal. Very useful.

The inactivation of restrictive enzymes depend on your vector size. If the vector size is more than 5kb, you need to inactivate your enzymes before ligation, because these enzymes hinder the transformation and you will get few number of clones. If your vector size is smaller, then its ok, you can use without inactivation and you will get enough clones for plasmid expansion

Hi Mahbobeh, 

I do vector or DNA insert restriction digests and do not bother to heat inactivate them. Instead, I simply load my digested samples in an agarose gel and then cut and gel purify the fragments I want, followed by ligation reactions. I don't usually have any problems using this method. If you were not going to gel purify your DNA after restriction digestion, that's when I would recommend that you should heat inactivate the enzymes, otherwise I would say that it is unnecessary because you are going to purify the DNA anyway. 

Good luck with your cloning! 

If you are carrying out a gel purification of your bands prior to ligation, there is no need to heat inactivate.

In my experience, heat inactivation results in reduced ligation efficiency further down the line. Avoid if possible.

thanks alot.

Mangal Singh

If the digested fragments are loaded on the agarose gel and purified, the heat innactivation is not necessary .