|EN31-005||500 U ( 2 U/μl )||Ask for an offer|
|EN31-025||2500 U ( 2 U/μl )||Ask for an offer|
Masterase is a 43.3 kDa heat-labile recombinant endonuclease, derived from a cold water eukaryotic organism, expressed in Pichia pastoris. The enzyme displays high specific activity towards double-stranded DNA leaving single-stranded DNA or RNA undamaged in standard conditions.
Masterase can be easily inactivated by heat treatment in moderate temperatures. It is intended for applications where the presence of dsDNA influences experiments’ results in thermo-sensitive applications and it is extremely useful for rapid and safe purification of RNA or proteins samples from contaminating DNA.
The enzyme hydrolyzes phosphodiester linkages yielding oligonucleotides with a 5′-phosphate and a 3′-hydroxyl groups.
Features and advantages
- Highly active in a broad temperature range (10 – 47°C).
- Low-temperature activity to protect RNA or proteins.
- Highly active in typical buffer formulations and broad pH range (optimum at 7.0 – 8.0).
- Requires at least 2 mM Mg2+ ions for optimal activity.
- Irreversible inactivation at low temperature (15 min at 52°C, 1 mM DTT) to secure biological product’s integrity.
- The activity towards dsDNA is at least 1000 times higher than towards ssDNA.
- dsDNA digestion (plasmid DNA, genomic DNA, etc.).
- RNA and protein samples rapid purification.
- PCR, qPCR Master Mixes, or other diagnostic reagents decontamination.
- Degradation of DNA template in transcription reactions.
One unit (U) is defined as the amount of enzyme that causes an increase in absorbance at 260 nm of 1.0 in 30 minutes at 37°C in 50 mM Tris-HCl buffer, pH 8.0 (25°C) supplemented with 5 mM MgCl2, 0.1 mg/ml BSA and 0.5 mg/ml herring sperm DNA as a substrate.
- The optimal final concentration of Masterase in a reaction depends on several factors (level of nucleic acids contamination, compounds present in the reaction mixture, temperature and time of incubation).
- For a sample with high DNA content we recommend using 0.5 – 1.0 U Masterase per 20 μl reaction mixture and incubation at 37°C for 30 min.
- For the optimal Masterase activity Mg2+ ions are required (2 – 4 mM optimally).
- Presence of Ca2+ ions (1 – 5 mM optimally) increases the activity of Masterase.
- Inactivation of Masterase depends on the concentration of the reducing agent, inactivation time and temperature. The enzyme requires at least 1 mM DTT or TCEP to be completely inactivated. We recommend inactivating Masterase by incubation at 52°C for 15 min in the presence of reducing agents such as DTT or TCEP (1 – 10 mM).
- There is no need to physically or chemically remove the Masterase from the reaction, before downstream processing, as heat treatment completely and irreversibly removes its activity.
Tolerance to most popular purification buffers’ additives:
|Variable/Parameter||Activity Range||Optimum Ativity Range|
|pH||6.0 – 10.0||7.0 – 8.0|
|Temperature||4 – 47°C||10 – 47°C|
|Mg2+||0 – 50 mM (Ca2+ ions increases the activity)||2 – 4 mM|
|Ca2+||0 – 100 mM||1 – 5 mM|
|Ammonium sulfate||0 – 100 mM||0 – 50 mM|
|NaCl / KCl||0 – 250 mM||0 – 100 mM|
|Imidazole||0 – 400 mM||0 – 300 mM|
|Urea||0 – 2 M||0 – 1 M|
|Glycerol||0 – 50%||0 – 40%|
|Triton X-100||0 – 2%||0 – 2%|
|DTT (at low temp.)||0 – 100 mM||0 – 100 mM|
|β-mercaptoethanol||0 – 2.5%||0 – 1%|
20 mM Tris-HCl, pH 8.0; 20 mM NaCl; 2 mM MgCl2; 1 mM CaCl2; 50% (v/v) glycerol
Store at -20°C in a freezer without a defrost cycle.
For long-term storage place at -80°C freezer.
Masterase is stable at -20°C for at least 3 years from the date of product release.
No loss in activity was observed after 6 months of incubation at RT.
Masterase does not lose its activity at least fifteen successive freeze/thaw cycles.
Shipping at room temperature or on blue/dry ice.
- The purity >95% determined by densitometry of SDS-PAGE SDS-PAGE
- Undetectable RNase activity after incubation of 5 U Masterase with 1 μg of RNA for 1 hour at 37°C.
- Undetectable proteolytic activity.