What is the Conditions and requirements that influence Enzyme Activity for the use of Restriction Endonuclease at the time of treatment with plasmid ?

What is the Conditions and requirements that influence Enzyme Activity for the use of Restriction Endonuclease at the time of treatment with plasmid ?

One unit activity :-

it is defined as amount of enzyme required to digest 1µg of reference DNA in 60 minutes at 370C. Usually reference DNA is λ phage DNA.

Buffer:- 

The most critical determinant of the enzyme activity is the ionic concentration (NaCl content) of the buffer. There are commercially 3 buffer systems available for the activity, low salt buffer (20mM), medium (100mM) or high (250mM) NaCl buffers. The buffer is usually 10mM Tris-HCl, pH 8.0, supplemented with Magnesium salt (often 50mM MgCl2), a reducing agent (usually 1mM DTT), and some enzymes require BSA (100µg/ml) and salt (NaCl). The reaction does not require ATP.

Quality of DNA :-

Preparation of DNA that has to be cleaved should be free of contaminants such as phenol, CHCl3, alcohol, EDTA, detergents, excessive salts, protein, RNA, all of which can pose a hindrance to the enzyme’s activity.

Stopping a Reaction :

To terminate Restriction enzymes activity, heat inactivation is a way but for the enzymes which it does not work, phenol / CHCl3 extraction is another means of inactivation of Restriction enzymes.

Incubation Temperature and Time  : - 

Recommended temperature for most of the Restriction enzymes is 370C except for the ones which are isolated from thermophilic bacteria which require higher temperature ranging from 50-650C. While some restriction enzymes have short ½ lives at 370C and for those lower temperatures are required. Incubation time as per the unit definition is 1 hour but it can be manipulated. Example, it may be shortened if more units of Restriction enzyme is added or longer times are used to allow a reaction to proceed to completion with fewer enzymes units.