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Reaction Conditions for Restriction Enzymes:

• General Protocol for DNA Digestion
• Five Buffer System
• Dilution of Restriction Enzymes
• Stability During Prolonged Incubation
• Inactivation (see below)
• Considerations for Partial Digestion of DNA
• Chart: "Reaction Conditions for Restriction Enzymes"

Inactivation

Before subsequent manipulation of the digested DNA, restriction enzymes present in the reaction mixture should be inactivated or removed.
Thermal inactivation of restriction enzymes is the most convenient method for terminating the digestion reaction. Most restriction enzymes can be heat-inactivated at 65°C or 80°C in 20 min. Information on the susceptibility of Fermentas restriction enzymes to thermal inactivation is presented in the table "Reaction Conditions for Restriction Enzymes", in the product descriptions and in the Certificate of Analysis supplied with each enzyme.
An alternative method to stop the reaction is by the addition of EDTA, which chelates Mg²⁺, thereby preventing DNA digestion. The recommended final concentration of EDTA is 20 mM.
However, high EDTA concentration is not compatible with most of downstream applications. Therefore, we recommend purification of the digested DNA with our DNA Gel Extraction Kit (#K0513) or phenol/chloroform extraction using the protocol below.

Note  
BfiI is the only known restriction enzyme that does not require Mg²⁺ for DNA cleavage. Therefore a digestion reaction catalyzed by this enzyme can not be terminated by the addition of EDTA. The enzyme can be inactivated by heating at 65°C for 20 min.

Protocol for DNA Purification after Enzymatic Reaction by Phenol/chloroform Extraction and Alcohol Precipitation

1. Mix your sample with 0.5 volume of TE-saturated phenol and 0.5 volume of chloroform. Then, centrifuge (10,000 rpm, 5 min, room temperature).
2. Transfer the upper phase to a fresh tube. Add an equal volume of chloroform and mix. Then, centrifuge (10,000 rpm, 5 min, room temperature).
3. Transfer the upper phase to a fresh tube. Add 1/10 volume of 3 M sodium acetate or 2 M sodium chloride.
4. Add an equal volume of isopropanol or 2.5 volumes of ethanol to precipitate DNA.
5. Incubate the mixture for 30-60 min at -20°C.
6. Centrifuge for 10 min at 10,000 rpm. Then discard the supernatant and rinse the pellet twice with 70% cold ethanol.
7. Air-dry the pellet. Dissolve in water, nuclease-free or TE buffer for further use.

Note
Use Glycogen to maximize the yield of DNA during precipitation, see detailed protocol.

Considerations for Partial Digestion of DNA

In certain cloning experiments, incomplete cleavage of the DNA is desirable. Such partial digestion of the DNA requires the following conditions:

• low amounts of 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 (see Site Preferences by Restriction Enzymes).

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Updated vasario 01, 2008 09:29