Description
dsDNase HL (500 U) 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. It is supplied with 20 mM Tris-HCl, pH 8.0; 20 mM NaCl; 2 mM MgCl 2; 1 mM CaCl 2; 50% (v/v) glycerol. One unit 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 MgCl 2, 0.1 mg/mL BSA and 0.5 mg/mL herring sperm DNA as a substrate.
Assay Specification Protein Purity >90% RNase activity None detected Proteolytic activity None detected
dsDNase HL can be quickly inactivated by heat treatment in moderate temperatures. It is intended for applications where the presence of dsDNA influences experiments’ results in thermo-sensitive applications. It is also beneficial for the rapid and safe purification of RNA or protein samples from contaminating DNA. The enzyme hydrolyzes phosphodiester linkages yielding oligonucleotides with a 5′-phosphate and 3′-hydroxyl groups.
Quality Control Protein concentration was determined by densitometry of SDS-PAGE electrophoresis using Coomassie Blue detection assay. The presence of RNase activity was determined after incubation of 5 U dsDNase HL with 1 μg of total eukaryotic RNA in a 20 μL volume for 1 hour at 37°C. The presence of proteolytic activity was determined by SDS-PAGE with Coomassie Blue detection following incubation of 1 μg of BSA with 100 U of enzyme for 20 hours at 37°C. A 1.575 mL reaction volume containing herring sperm DNA as a substrate is incubated for 30 minutes at pH 8 at 37°C with an enzyme that degrades DNA to acid-soluble oligonucleotides. One unit of the enzyme causes an increase in absorbance at A260 of 1.0.
