1. Lysozyme
Prepare a 50 mg/ml lysozyme solution with water, divide into aliquots and store at -20°C. Discard each aliquot after use.
2. Proteolytic enzymes
| Storage solution | Storage temperature | Reaction concentration | Reaction buffer | temperature | Preprocessing | |
| 0.01mol/L Tris (pH7.8) | ||||||
| Pronase A | 20mg/ml | -20℃ (soluble in water) | 1mg/ml | 0.01mol/L EDTA | 37℃ | Autodigestion b |
| 0.5% SDS | ||||||
| 0.01mol/L Tris (pH7.8) | ||||||
| Proteinase K c | 20mg/ml | -20℃ (soluble in water) | 50μg/ml | 0.005mol/L EDTA | 37～56℃ | No pre-processing required |
| 0.5% SDS | ||||||
a: Pronase is a mixture of serine enzyme and acid protease isolated from Streptomyces griseus.
b: Self-digestion can eliminate the contamination of DNA and RNA enzymes. The preparation method of self-digested pronase is as follows: dissolve the enzyme powder in 10mmol./l Tris·HCl (pH7.5), 10mmol/l NaCl to a concentration of 20mg/ml, and incubate at 37℃ for 1h. Digested pronase is divided into small portions and placed in sealed test tubes and stored at -20℃.
c: Proteinase K is a highly active protease of the subtilisin class, purified from Tritirachium album Limber. The enzyme has two Ca 2+ binding sites that are some distance from the active center of the enzyme and are not directly related to the catalytic mechanism. However, if Ca 2+ is removed from the enzyme, the catalytic activity will be lost by about 80% due to long-range structural changes, but the remaining activity is usually sufficient to degrade proteins that normally contaminate acid products. Therefore, EDTA is usually added during proteinase K digestion (to inhibit the action of Mg 2+ dependent nucleases). However, if proteins that are highly resistant to proteinase K are to be digested, such as keratins, it may be necessary to use a buffer containing 1mmol/l Ca 2+ but no EDTA. EGT (pH 8.0) should be added to a final concentration of 2mmol/L after digestion and before purification of nucleic acids to chelate Ca 2+.
3. RNase without DNase
Dissolve pancreatic RNase (RNase A) in 10 mmol/l Tris·HCl (pH 7.5) and 15 mmol/l NaCl to a concentration of 10 mg/ml, heat at 100°C for 15 min, slowly cool to room temperature, and divide into small portions and store at -20°C.
