Restriction enzymes are endonucleases that recognize specific double stranded DNA sequences and cleaves the DNA in both strands. Restriction enzymes used in cloning are site specific. Cloning involves cleaving both the vector and genomic DNAs with a restriction endonuclease which yields compatible sticky ends, and then using those cohesive ends to recombine the DNAs into a construct or recombinant molecule. The vector is cleaved in one location, while the genomic DNA is extensively digested into a pool of fragments. The quality of restriction enzyme determines the successful of cloning process. Therefore, its storage is very important to be known by us.
The stability of preparations of purified restriction enzymes critically depends on adequate storage conditions, in particular, on buffer composition, enzyme concentration, presence of additives, and an appropriate storage temperature.
Different buffer compositions are recommended for the storage of different enzymes. The buffer composition for most restriction enzymes are :
- 10-50 mM Tris-HCl pH 7.5
- A minimum of 50 mM NaCl or KCl
- 1 mM EDTA, and
- 60% (v/v) glycerol
That composition has proven to be optimal. Many restriction enzymes require additional additives, most commonly:
- 1 mM 1,4-dithiothreitol
- 1,4-dithioerythritol or beta-mercaptoethanol, its function is to prevent oxidation of cysteine residues
- 0.01-0.1% (w/v) Triton X-100, Tween, Lubrol, or other detergents
- As well as 0.1 mg/mL nuclease-free bovine serum albumin, this component is to prevent aggregation and precipitation.
Restriction enzymes should be stored in unfrozen solution at temperatures below 0oC preferably at -20oC in a 60% glycerol solution. If stored frozen at -70oC repeated thawing and freezing should be avoided since the heat generated during the defrost cycles of frost-free freezers may adversely affect the stability of a restriction enzyme preparation.