GENERAL USE OF ENZYMES IN MOLECULAR BIOLOGY
Things you should know about restriction enzymes:
- They are heat-sensitive and must be stored at -20oC; keep
them on ice while you remove enzyme from the vial.
- 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.
- The enzymes that we purchase are packaged such that 1 µl of enzyme
is sufficient to digest 10 -12 µg of DNA. Therefore, you only need 1
µl of enzyme. If you add too much, the enzyme may cut nonspecifically.
- 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.
- The enzymes work best at certain temperatures (usually 37oC
- check the catalog or product information).
Sample digestion reaction:
10 µl chromosomal DNA
3 µl buffer
16 µl nanopure water
1 µl EcoR I (ADD THIS LAST)
30 µl total digest
Incubate O/N at 37ûC
Things you should know about T4 ligase:
- 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.
- 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.
- The buffer is supplied at 10X concentration; it must be diluted
- 1 µl of enzyme is sufficient to ligate 10 -12 µg of DNA. Therefore,
you normally only need 1 µl of enzyme.
- 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.
- Ligase works best at 15oC. We run our ligation reactions
in the refrigerated centrifuge.