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Reaction Conditions for Restriction Enzymes:

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

We recommend digesting DNA with a 2-fold to 10-fold excess of enzyme in the total volume of 20 µl using 0.2-1.5 µg of DNA. A typical restriction enzyme digestion protocol is presented below.

General Protocol for DNA Digestion

1. Add components in the following order:
   

   Water, nuclease-free	16 µl
   10X recommended buffer for restriction enzyme	2 µl
   Substrate DNA	1 µl (~1 µg)
   Restriction enzyme	0.5-1 µl (5-10 u)

2. Mix gently, spin down briefly.
3. Incubate at the optimum temperature for 1-16 hours.

The digestion reaction may be scaled either up or down.

Note
Some enzymes require additional components to obtain the stated activity. In these cases, add the required additive and adjust the volume of water appropriately.

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Updated gegužės 09, 2007 16:38