Summary: Molecular Biology Basics
Planning Restriction Enzyme Digests
A. Checklist: Buffer type
Addition of BSA
Number of units of enzyme
B. Plan to digest DNA with an "excess" of enzyme activity. Plan for the "excess" to be divided
between time of digestion and number of units of enzyme activity. For clonings, plan carefully
because excess disgestion may result in damage to DNA ends. Parameters to consider:
Amount and type of DNA: estimate mass and note type (circular, linear, etc).
Number of units: check relative efficiency of digestion of different substrates (circular, linear, etc).
Time of digestion: check survival of restriction enzyme(s) in digestion.
Overnight digests: plan for the minimum number of units necessary.
Star Activity: determine if enzyme is sensitive to high glycerol, low salt, etc.
C. For digestions at 50-65°C, consider loss of volume due to evaporation. Do a short digestion, use a
digestion volume 20 µl, incubate in convection incubator, or cover reaction with minearl oil.
D. For double digests, consider: buffer selection, addition of BSA, proximity of sites. For double
digests with incompatible enzymes: digest with first enzyme, extract with phenol (see below),
ethanol precipitate and wash (see below), and digest with second enzyme.
E. Inactivation: chelate Mg++