|EN33-050||5000 U ( 20U/μl )||Ask for an offer|
|EN33-250||25000 U ( 20U/μl )||Ask for an offer|
DNAseMe dsDNase (EN33)
DNaseMe is a 42.8 kDa recombinant endonuclease, derived from marine amphipods, expressed in Pichia pastoris. The enzyme displays high specific activity towards double-stranded DNA leaving single-stranded DNA or RNA undamaged in standard conditions. DNaseMe is highly active in a broad spectrum of temperatures, buffer conditions and pH. The specific activity is similar to bovine DNase I however, DNaseMe is characterized by higher stability in demanding reaction and storage conditions (e.g. high salt and detergent containing buffers, elevated temperature).
These features make DNaseMe extremely useful for rapid and “RNA safe” degradation of genomic DNA, where absence of ribonucleases is critical to maintain the integrity of RNA.
The enzyme hydrolyzes phosphodiester linkages yielding oligonucleotides with a 5′-phosphate and a 3′-hydroxyl groups.
Features and advantages
- Active in a broad temperature range (10 – 80°C).
- Active in a broad pH range (optimum at pH 6.0 – 9.0).
- Highly active at elevated salt concentrations and other typical buffer additives (Table 1), which significantly improves efficiency and yield of various workflows.
- Requires bivalent cations (Mg2+ and Ca2+) for maximal activity.
- Degrades dsDNA to fragments below 10 nt.
- The activity towards dsDNA is at least 1000 times higher than towards ssDNA.
- Extraction and purification of RNA (equivalent of DNase I).
- Removal of contaminating genomic DNA from RNA samples.
- Degradation of DNA template in transcription reactions.
- Reduction of viscosity in biological samples.
- Removal of residual DNA during primary stem cell isolation, biopharma and bioprocessing procedures.
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 and pH 8.0 with herring sperm DNA as a substrate.
- The optimal concentration of DNaseMe in the final reaction mixture depends on several factors (level of nucleic acids contamination, temperature and time of incubation, salt concentration and other compounds present in the reaction mixture). The amount of DNaseMe and incubation conditions have to be determined experimentally (we recommend using 35 U DNaseMe for digestion of up to 50 μg dsDNA at 37°C for 10 – 15 min).
- In the presence of Mg2+ or Ca2+ ions, DNaseMe shows high endonuclease activity towards dsDNA leaving ssDNA and RNA essentially intact.
- Presence of Ca2+ ions increases the total activity of DNaseMe.
- In the presence of Mn2+ ions, the enzyme shows slightly elevated DNaseMe activity including activity towards ssDNA and RNA. Avoid using Mn2+ ions during purification of RNA or ssDNA.
- Inactivation of DNaseMe depends on the concentration of the reducing agent, inactivation time and temperature. We recommend inactivating DNaseMe by incubation at 85°C for 15 min in the presence of reducing agents such as DTT or TCEP (1 – 10 mM).
- The enzyme requires 1 – 10 mM DTT or TCEP to be completely inactivated.
- Alternatively, DNaseMe can be inactivated and removed by using a spin column or phenol/chloroform extraction.
Tolerance to most popular purification buffers’ additives:
|Variable / Parameter||Activity Range|
|pH||4.0 – 9.5 (optimum 6.0 – 9.0)|
|Temperature||10 – 80°C|
|Inactivation||85°C/15 min/1 mM DTT|
|Mg2+||1 – 150 mM (Ca2+ increases the activity)|
|Ca2+||1 – 50 mM|
|Ammonium sulfate||0 – 0.5 M|
|NaCl / KCl||0 – 2 M|
|Imidazole||0 – 0.4 M|
|Urea||0 – 4 M|
|Glycerol||0 – 50%|
|Triton X-100||0 – 20%|
|SDS||0 – 0.5% (not recommended)|
|DTT (at low temp.)||0 – 100 mM|
|β-mercaptoethanol||0 – 2.5%|
20 mM Tris-HCl, pH 8.0; 50 mM KCl; 5 mM MgCl2; 50% (v/v) glycerol
Store at -20°C in a freezer without a defrost cycle. For long-term storage place at -80°C freezer.
DNaseMe is stable at -20°C for at least 3 years from the date of product release.
No loss in activity was observed after 12 months of incubation at RT.
No loss in activity was observed after 1 hour of incubation at 75°C.
DNaseMe does not lose its activity at least fifteen successive freeze/thaw cycles.
Shipping at ambient temperature or on blue ice.
- The purity >95% determined by densitometry of SDS-PAGE.
- Undetectable RNase activity after incubation of 10 U dsDNase with 1 μg of RNA for 1 hour at 37°C.
- Undetectable proteolytic activity.