Partial Digestion of DNA

Certain cloning experiments may require incomplete DNA cleavage. Such partial digestion of the DNA can be achieved by using the following conditions:

• suboptimal concentration of the restriction enzyme in the reaction mixture;
• short incubation time;
• incubation at a suboptimal temperature.

For certain targets, partial cleavage of the desired DNA site is inefficient due to site preferences of restriction enzymes.

Inactivation of Restriction Enzymes

Inactivation of restriction enzymes following a digestion reaction is often required for downstream applications.
Thermal inactivation is a convenient method used to terminate enzyme activity. The majority of restriction enzymes can be heat-inactivated at 65°C or 80°C in 20 min. Information on susceptibility of Fermentas restriction enzymes to thermal inactivation is listed in the Table “Reaction Conditions for Restriction Enzymes”, as well as in product descriptions and Certificates of Analysis.
All known restriction enzymes with exception of BfiI require Mg²⁺ for DNA cleavage. Thus, addition of EDTA (20 mM final concentration) to the reaction mixture is an alternative method that can be used to halt digestion. EDTA is generally not compatible with most of downstream applications, therefore purification of the digested DNA with a spin-column or silica powder (#K0513) based kit or by chloroform extraction is recommended.

Dilution of Restriction Enzymes

Dilution Buffer for Restriction Enzymes (#B19) is available from Fermentas for applications that require diluted enzymes.
Enzymes diluted in this buffer retain 50-100% activity after storage for one month at -20°C.