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GENERAL USE OF ENZYMES IN MOLECULAR BIOLOGY

Restriction Enzymes

Things you should know about restriction enzymes:

  1. They are heat-sensitive and must be stored at -20oC; keep them on ice while you remove enzyme from the vial.
  2. They work best in a specified salt concentration. The enzymes that we purchase come with the correct buffer but these buffers are at 10X concentration; they must be diluted 10-fold. If you are digesting DNA with more than one restriction enzyme at a time, it is important to check for the compatibility of the buffers. If the enzymes do not use the same buffer you can usually "get away with" using a combination of the buffers as long as they are not drastically different. If the buffers are very different, it is better to do one digestion at a time and to precipitate and re-suspend the DNA between digestions.
  3. The enzymes that we purchase are packaged such that 1 l of enzyme is sufficient to digest 10 -12 g of DNA. Therefore, you only need 1 l of enzyme. If you add too much, the enzyme may cut nonspecifically.
  4. The enzymes that we purchase are diluted in a glycerol solution to prevent freezing at -20oC. The glycerol can interfere with digestion so you will need to dilute the enzyme at least 1:10 in the total digestion solution.
  5. The enzymes work best at certain temperatures (usually 37oC - check the catalog or product information).

Sample digestion reaction:

10 l chromosomal DNA
3 l buffer
16 l nanopure water
1 l EcoR I (ADD THIS LAST)
30 l total digest

Incubate O/N at 37ûC

T4 Ligase

Things you should know about T4 ligase:

  1. Both the enzyme and its buffer are heat-sensitive and must be stored at -20oC; keep them on ice while you remove enzyme from the vial. The buffer is heat sensitive because it contains ATP.
  2. The ligase is sensitive to any foreign proteins or salts in the reaction mix and therefore you should clean your DNA by precipitation or other method before setting up a ligation reaction.
  3. The buffer is supplied at 10X concentration; it must be diluted 10-fold.
  4. 1 l of enzyme is sufficient to ligate 10 -12 g of DNA. Therefore, you normally only need 1 l of enzyme.
  5. The enzyme is diluted in a glycerol solution to prevent freezing at -20oC. As with restriction enzymes, the glycerol can interfere with ligation so you will need to dilute the enzyme at least 1:10 in the total ligation reaction.
  6. Ligase works best at 15oC. We run our ligation reactions in the refrigerated centrifuge.