Enzymatic cleanup | Removing unwanted residues and artifacts

These good housekeepers polish, snip and trim

Reagents and reactants that are left behind can hinder downstream processes. Use the right enzyme preparation to clean up your samples before taking the next step. Speed up and enhance amplification and transcription with some not-so-secret additives.

Enzymes for cleavage and cleanup of residual reagents

Enzymatic cleanup of DNA samples with Exonuclease I, DNase I, RNase A, Proteinase K and other nucleases and proteases removes residual primers, nucleotides and enzymes prior to SNP analysis, next-generation sequencing, Sanger DNA sequencing, bioprocessing or other downstream procedures.

Proteinase K is a broad-spectrum endopeptidase widely used for the digestion of proteins, including DNases and RNases. A digestion step with the enzyme is routinely applied during nucleic acid preparation without affecting the integrity of isolated DNA or RNA. Proteinase K is active under a wide range of reaction conditions, including elevated temperatures and the presence of SDS.

Proteinase K NGS Grade is available for the most demanding applications, including preparation of nucleic acids for next-generation sequencing. Extensive purification yields a high-quality enzyme product with increased specific activity, significantly increased solubility (2.5 fold) and remarkable purity with DNA content ≤0.1 pg/mg. Proteinase K NGS Grade is free of exonucleases, endonucleases and ribonucleases.

The TAGZyme DAPase enzyme and TAGZyme system is used for His-tag removal from proteins containing an intrinsic DAPase stop point (expressed using the TAGZyme pQE-2 vector) or from proteins that contain an engineered glutamine stop point.

A comprehensive range of housekeeping enzymes is available for cleavage and cleanup of residual reagents.

Enzyme	Activity	Application
RP100B, RP101B, RP103B, RP107B Proteinase K (molecular biology grade)	BLIRT Proteinase K (MB/PCR grade) is a subtilisin-related serine protease isolated from Parengyodontium album (Tritirachium album)	Extraction of DNA and RNA from different starting materials. Removal of DNases and RNases during nucleic acid isolation. Purification of samples contaminated with different proteins. Recommended for automated isolation stations.
RP100N, RP101N, RP102N, RP103N Proteinase K Ultrapure (NGS grade)	BLIRT Proteinase K (Ultrapure/NGS grade) is a subtilisin-related serine protease isolated from Parengyodontium album (Tritirachium album); extensive purification yields the highest quality enzyme product	Demanding applications such as preparation of DNA for NGS libraries. Extraction of DNA and RNA from different starting materials. Removal of DNases and RNases during nucleic acid isolation. Purification of samples contaminated with different proteins Recommended for automated isolation stations
19131 Proteinase K	QIAGEN Proteinase K is a subtilisin-type protease isolated from the saprophytic fungus Tritirachium album	Protease digestion in DNA and RNA isolation procedures Proteinase K possesses a high specific activity; stable over a wide range of temperatures and pH values; not inhibited by EDTA; suitable for short digestion times
19155 Protease	QIAGEN Protease is a serine protease isolated from a recombinant Bacillus strain; an economical alternative to Proteinase K	Protease digestion in isolation of native DNA and RNA from a variety of sources
X8010L Exonuclease I Coming soon	Exonuclease I cleaves single-stranded DNA in the 3’→5’ direction, releasing 5’-mono/dinucleotides and leaving double-stranded DNA molecules and the 5’-terminus intact; digestion is inhibited by the presence of a 3’-terminal phosphate	Removing single-stranded primers in PCR reactions prior to SNP analysis or Sanger DNA sequencing Removing single-stranded primers for nested PCR reactions Removing linear single-stranded DNA, leaving behind double- stranded DNA in the sample
79254 RNase-free DNase	Endonuclease that nonspecifically cleaves DNA to release di-, tri- and oligonucleotide products with 5’-phosphorylated and 3’-hydroxylated ends; acts on ssDNA and dsDNA, chromatin and RNA:DNA hybrids	Removing contaminating DNA from RNA solutions prior to RNA cleanup and concentration Removing DNA from protein samples Nicking DNA as a first step to incorporating labeled bases into DNA DNA-protein interaction analysis (Footprinting assay)
EN31 dsDNase-HL BLIRT	Heat-labile endonuclease derived from a cold-water eukaryote; high specific activity towards dsDNA leaving ssDNA or RNA undamaged; easily inactivated by moderate heat treatment	Intended for thermo-sensitive applications where the presence of dsDNA influences results Rapid and safe purification of RNA or protein samples from contaminating DNA
EN33 dsDNase BLIRT	Endonuclease derived from marine amphipods; high specific activity towards dsDNA leaving ss DNA or RNA undamaged; highly active in a broad spectrum of temperatures, buffer conditions and pH	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 biopharma and bioprocessing procedures
EN32 Saltonase (HL-Nuclease) BLIRT	Cold-active, heat-labile endonuclease originating from psychrophilic bacteria; digests all DNA and RNA substrates in different buffer conditions and a broad range of temperatures, including low temperatures and environments with high salt content	Purification of biologics from residual nucleic acids in biopharma manufacturing Purification of recombinant proteins and enzymes for research use Removal of undesired nucleic acids contamination in molecular biology reagents in demanding systems Reduction of viscosity in biological samples (during production, automation)
RP145, 19101 RNase A	Endoribonuclease that degrades ssRNA at C and U residues	Removing contaminating RNA from plasmid and genomic DNA preparations Removing RNA from recombinant protein preparations Mapping single-base mutations in DNA or RNA (RNase protection assay)
34362 TAGzyme DAPase Enyzme	TAGZyme DAPase (Recombinant dipeptidyl peptidase I)	Removing dipeptides sequentially from N-terminal His tags up to the "stop point" expressed using TAGZyme pQE vectors Production of His-tag-free proteins for: Protein structure determination Therapeutic proteins

Improving yields

Improved PCR yields and quality of templates may be achieved with the use of proteins that counteract PCR inhibitors and by adding specialized DNA-binding proteins to amplification and sequencing reactions. The DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32), increases PCR amplification efficiency with a number of diverse templates. In addition, using E. coli single-stranded DNA-binding protein (SSB) in DNA sequencing reactions increases the resolution of sequencing runs.

An increased rate of in vitro transcription is made possible by treatment with inorganic pyrophosphatase, an essential component of reactions for RNA preparation. This enzyme cleaves pyrophosphate into two phosphate molecules and prevents pyrophosphate from precipitating with magnesium.

Reagent	Activity	Application
Y9030L E. coli ssDNA Binding Protein Coming soon	Binds with high specificity to single-stranded DNA; thermostable	Useful in primer sequestering Useful in enhancing the specificity of PCR Useful in enabling the sequencing of problematic DNA templates
Y9130L T4 Gene 32 Protein Coming soon	Stabilizes ssDNA regions	Increases processivity of some DNA polymerases
RP50, RP51 PCR Anti-Inhibitor	Mixture of alkaline proteins to counteract substances that inhibit PCR originating in DNA template isolation	Increases PCR yields with difficult DNA templates isolated from urine, saliva, sputum, blood, cell swabs, cerebrospinal fluid, biopsies, etc.
Y9380L E. coli pyrophosphatase Coming soon	An inorganic pyrophosphatase that catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate*	Increases RNA yield in transcription reaction Enhancing DNA replication
Y9370L Thermostable pyrophosphatase Coming soon	Thermostable pyrophosphatase is a recombinant enzyme from Sulfolobus acidocaldarius that catalyzes the hydrolysis of inorganic pyrophosphate to produce orthophosphate†	Useful for the enhancement of DNA replication in PCR

UDG strand cleavage

UDG-mediated strand cleavage is an important tool in molecular biotechnology, allowing for controlled and location-specific cleavage of single- and double-stranded DNA chemically or enzymatically synthesized with single or multiple incorporations of deoxyuridine.

UDG treatments can reduce sequence artifacts, eliminate PCR carry over and generate specific gaps in DNA.

Enzyme	Activity	Application
19160, G5010L, EN19 Uracil DNA Glycosylase (UDG) Coming soon	Uracil DNA glycosylase (UDG) is involved in base excision repair; UDG creates abasic sites in uracil-containing DNA; degrades uracil-containing DNA; does not excise uracil from RNA or oligonucleotides	DNA pretreatment with UDG reduces DNA artifacts during mutation detection by next-generation sequencing and other methods, without affecting capacity to detect real mutations Removes uracil (deaminated cytosine) artifacts from FFPE preps Thermolabile UDG can be used to eliminate PCR “carry over” contamination when dUTP is substituted for dTTP in the primary PCR reaction mix As a probe for protein-DNA interaction studies Glycosylase mediated single nucleotide polymorphism detection (GMPD)
G5020L Thermolabile UDG Coming soon	Thermolabile UDG creates abasic sites in uracil-containing DNA; degrades uracil-containing DNA; gene isolated from a marine bacterium; inactivated by incubation for 10 min at >50°C	Thermolabile UDG can be used to eliminate PCR “carry over” contamination when dUTP is substituted for dTTP in the primary PCR reaction mix
Y9180L 10X Uracil Cleavage System Coming soon	The system consists of two enzyme components, Uracil DNA Glycosylase and Endonuclease VIII. When the enzymes are added sequentially to a reaction where a synthetic DNA fragment contains a deoxyuracil, a single nucleotide gap is generated at the location of the uracil residue	Enzymatically generates uracil nucleotide gaps in vitro UDG is a DNA-specific enzyme that does not excise uracil from RNA substrates. Targeted incorporation of deoxyuridine nucleotides into oligonucleotides may be used with the enzyme system for the specific cleavage of sections of DNA, or DNA templates, from DNA:RNA hybrids.
Y9080L Endonuclease VIII Coming soon	Endonuclease VIII functions as both an N-glycosylase (by excising oxidative base lesions) and an AP lyase (by subsequently cleaving the phosphodiester backbone), leaving terminal phosphates at the 5’ and 3’ ends; participates in base excision repair of oxidatively generated DNA damage	Excises damaged pyrimidines from duplex DNA Used to measure DNA damage via single cell gel electrophoresis (Comet assay)

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