Qiagen, EN31-025, dsDNase HL (2500 U)

Concentration: ≥2 U/μL Shelf life: 36 months. Storage temperature: -20°C without a defrost cycle. For long-term storage, place at -80C.

Features

- Highly active in a broad temperature range (10–47°C)
- Yields low temperature activity to protect RNA or proteins
- Highly active in typical buffer formulations and has a wide pH range with an optimum of 7.0–8.0
- Requires at least 2 mM Mg 2+ ions for optimal activity
- Possesses irreversible inactivation at low temperature (15 minutes at 52°C, 1 mM DTT)

Product Details

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.

Performance

Assay: Specification
Protein Purity: >90%
RNase activity: None detected
Proteolytic activity: None detected

Principle

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.

Procedure

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.

Applications

- dsDNA digestion for plasmid and genomic DNA
- RNA and protein samples rapid purification
- PCR, qPCR master mixes and other diagnostic reagents decontamination
- Degradation of DNA template in transcription reactions

This is used for applications such as: