Modifying Enzymes

Overview:Proteinase K is a robust and versatile serine protease used extensively in molecular biology for the digestion of proteins in nucleic acid preparations. This solution, at a concentration of 20 mg/mL, is ideal for a wide range of applications including the preparation of DNA and RNA samples. The PCR grade quality ensures that the enzyme is free from contaminants that could interfere with downstream applications, making it highly suitable for sensitive techniques such as PCR.Product Details:ready-to-useConcentration: 20 mg/mLactive over a wide range of reaction conditionsAn elevation of the reaction temperature from 37°C to 50 - 60°C may increase the activity several timesQuality: PCR grade, ensuring high purity and no interference with PCR reactions.Applications: Suitable for DNA and RNA extractionRemoval of protein contaminantsPreparation of samples for various molecular biology techniques.Benefits:High Purity: The PCR grade quality guarantees minimal contamination, ensuring reliable results in sensitive applications. Versatility: Ideal for various molecular biology procedures, including sample preparation for PCR.Convenience: Ready-to-use solution at an optimal concentration for ease of use. Notes:The recommended working concentration of Proteinase K is 0.05 to 1 mg/mL. The activity of the enzyme is stimulated by 0.2 to 1% SDS and also by 1 to 4M ureaCa2+ protects Proteinase K against autolysis and increases the thermal stabilityStable over a wide pH range: 4.0 to 12.5, optimum pH 7.5 to 8.0Activity optimum: 50°C to 55°CRapid denaturation of enzyme occurs at temperatures above 65°CFeatures:No detected exonuclease, endonuclease or RNase activitySpecific activity: >40 units/mg protein Activity: ≥30 units/mg lyophilizateSolubility: ≥20 mg/mLDNA content ≤10 pg/mgShipping conditions: Shipment on wet iceProteinase K can be obtained from Genaxxon as Powder (M3036) > or as 20mg/mL Solution (M3037) >.Produced by Genaxxon bioscience GmbH, founded in 2002 by Dr. Norbert Tröndle to provide reliable products for PCR and custom cell culture media formulations, the Proteinase K Solution 20 mg/mL offers a dependable and efficient solution for your molecular biology needs. Achieve consistent, high-quality results with an enzyme designed specifically for your requirements.

Overview:Proteinase K is a robust and versatile serine protease used extensively in molecular biology for the digestion of proteins in nucleic acid preparations. This solution, at a concentration of 20 mg/mL, is ideal for a wide range of applications including the preparation of DNA and RNA samples. The PCR grade quality ensures that the enzyme is free from contaminants that could interfere with downstream applications, making it highly suitable for sensitive techniques such as PCR.Product Details:ready-to-useConcentration: 20 mg/mLactive over a wide range of reaction conditionsAn elevation of the reaction temperature from 37°C to 50 - 60°C may increase the activity several timesQuality: PCR grade, ensuring high purity and no interference with PCR reactions.Applications: Suitable for DNA and RNA extractionRemoval of protein contaminantsPreparation of samples for various molecular biology techniques.Benefits:High Purity: The PCR grade quality guarantees minimal contamination, ensuring reliable results in sensitive applications. Versatility: Ideal for various molecular biology procedures, including sample preparation for PCR.Convenience: Ready-to-use solution at an optimal concentration for ease of use. Notes:The recommended working concentration of Proteinase K is 0.05 to 1 mg/mL. The activity of the enzyme is stimulated by 0.2 to 1% SDS and also by 1 to 4M ureaCa2+ protects Proteinase K against autolysis and increases the thermal stabilityStable over a wide pH range: 4.0 to 12.5, optimum pH 7.5 to 8.0Activity optimum: 50°C to 55°CRapid denaturation of enzyme occurs at temperatures above 65°CFeatures:No detected exonuclease, endonuclease or RNase activityConcentration ≥20 mg/mL / Activity ≥800 U/mLDNA content ≤200 pg/mLShipping conditions: Shipment on wet ice Storage buffer: 10 mM Tris/HCl, pH7.5; 1mM (CH3COO)2Ca; 50% glycerolProteinase K can be obtained from Genaxxon as Powder (M3036) > or as 20mg/mL Solution (M3037) >.Produced by Genaxxon bioscience GmbH, founded in 2002 by Dr. Norbert Tröndle to provide reliable products for PCR and custom cell culture media formulations, the Proteinase K Solution 20 mg/mL offers a dependable and efficient solution for your molecular biology needs. Achieve consistent, high-quality results with an enzyme designed specifically for your requirements.

Genaxxon Proteinase Low-Temp is a nonspecific endopeptidase derived from an Arctic marine microbial source. It has a broad substrate specificity and is easy to inactivate after use. Starting at a temperature of 60°C, the Genaxxon Proteinase Low-Temp is heat-inactivated, making it ideal for heat-sensitive samples. The Proteinase Low-Temp is most active in the temperature range of 25°C-40°C. Recommended inactivation conditions are: Typically up to 15 minutes at 40°C – 65°C (inactivation by denaturation). It is known that histones and other proteins shield nucleic acids from an optimal interaction with other DNA-binding proteins and enzymes. Genaxxon Proteinase Low-Temp is ideal for transforming chromatin and other compact nucleic acids into mere DNA. The enzyme is easily inactivated. This allows thermal inactivation at temperatures that provide RNA integrity and avoid the dissociation of dsDNA.Advantages:- Easy to deactivate- Active at high salt content- Compatible with downstream analyzes Application examples:Preparation of chromosomal DNA for PFGE- Degradation of nucleases (DNase, RNase) in the preparation of nucleic acids, including the recovery of native RNA- Isolation of genomic DNA from tissue of mouse tails- Isolation of genomic DNA from cell culturesSpecific activity: >30 U/mg. Foreign activity: RNase and DNase undetectable. Temperature optimum: + 25°C-40°C (pH 7-10). Recommended inactivation conditions: Typically up to 15 minutes at 40°C – 65°C (inactivation by denaturation).Inhibitors: General Serine protease inhibitors such as PMSF (M3194) >.Instructions for use: The enzyme is stable up to 2 years when stored at -20°C. Genaxxon Proteinase Low-Temp is stable even when stored at 4°C (>6 months) and room temperature (>4 weeks).The optimal enzyme activity is between pH 7 and 10. Recommended temperature range for the activity is 25-40°C. The activity of Genaxxon Proteinase Low-Temp is not dependent on divalent cations such as calcium (Ca2 +) and is therefore active in buffers containing EDTA (<40°C reaction temperature).Genaxxon Proteinase Low-Temp is suitable for the digestion of proteins in the presence of SDS (0.2-1%) and urea (1-5 M). The enzyme tolerates 500mM guanidine thiocyanate and 1% Triton X-100 (>50% activity). There is no significant loss of activity at 400 mM NaCl. Proteinase K is used for the digestion of proteins in cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively inactivates DNases and RNases. The digest with Proteinase K for the purification of nucleic acids is performed in the presence of EDTA (inhibition of magnesium-dependent enzymes). Proteinase K can be obtained from Genaxxon as Powder (M3036) > or as 20mg/mL Solution (M3037) >.

DescriptionRNase-free DNAse I is mainly used to remove DNA from RNA preparations. E.g. for experiments like RT-PCR, RT-qPCR or other applications that should be free of DNA. Since no purification method for RNA removes 100% of the DNA, it is advisable to remove DNA using DNAse I prior to any RT-PCR. A simple 15-minute digestion at room temperature reliably removes all contaminating DNA. DNase I is inactivated by adding the stop solution (1µL of a 50mM EDTA solution to 10µL of the DNA digest solution) and subsequent heating. Since heat also denatures RNA, the batch can then be used immediately for reverse transcription. Our RNase-free DNAse I is a recombinantly derived DNase I. Therefore, there are no problems with possible traces of animal origin.

DNase I is an endonuclease isolated from bovine pancreas that digests double- and single-stranded DNA into oligo- and mono-nucleotides. DescriptionDNAse I is used, among other things, to avoid unwanted cell clumping during tissue disaggregation, which makes dispersion of individual cells difficult. Since tissue disaggregation and subsequent isolation of single cells is always accompanied by rupture and lysis of some cells, DNA is released from these cells into the surrounding medium. The released DNA in the medium is thought to be responsible for undesirable cell clumping during tissue dissociation procedures. This clumping effect can be avoided by adding deoxyribonuclease (DNase) to the dissociation medium and has been described for a variety of different cell and tissue types. DNase I is suitable for eliminating DNA from RNA preparations prior to sensitive applications, such as RT-PCR. Since no RNA purification procedure removes 100% of the DNA, RNA samples should be digested with DNase I before RT-PCR. A simple 15 minute digestion at room temperature removes the contaminating DNA. DNase I is inactivated by adding the stop solution and heating. Heating also denatures the RNA, so the RNA can be used directly for reverse transcription. This DNAse I is not RNase-free and has to be chemically treated if used for RNA isolation procedures.

Ribonuclease A- DNase-free is a DNase and Protease-free enzyme that can be used directly for RNA digests, e.g. in DNA purification procedures. Elimination of potential DNAse activity is not necessary. Potential DNAse activities have been removed. DescriptionRibonuclease A is an endo-ribonuclease specifically cleaving single stranded RNA at the 3' side of pyrimidine bases (cytosine and uracil). RNAse A ist used to prepare RNA-free DNA, to digest non-hybridised regions of RNA-DNA hybrids and as a molecular weight marker. The pH optimum for RNAse A is between 7.0-7.5. RNAse A is inhibited by Diethylpyrocarbonate (DEPC) >, guanidinium salt (4M GuaSCN), beta-Mercaptoethanol, heavy metals, vanadyl-ribonucleoside complexes, RNAse-inhibitor from human placenta and by competitive DNA. RNAse A cleaves single and douple stranded RNA and RNA in RNA:DNA hybrids at low salt conditions (100mM NaCl). At high salt conditions (>300 mM NaCl), RNAse A cleaves single-stranded RNA only. The enzyme can only be removed by Proteinase K digest and subsequent phenol extraction. Stock solution: Stock solutions are prepared at concentrations from 1 - 10mg/mL in 10mM Tris/HCl, pH7.5; 15mM NaCl or in 10mM Tris/HCl, pH7.5; 1mM EDTA, pH8.0 (TE buffer).The recommended working concentration is 10µg/mL (removal of RNA from plasmid preparations; 1hr, RT) or 100ng/mL (preparation of 'blunt ends' of double-stranded cDNA). Stability:RNase A aggregates during lyophilizing and storage. It has a high affinity to glas surfaces, which has to be taken into consideration. At neutral pH (e. g. in PBS pH 7.4) and high concentrations (>10mg/mL) the enzyme will precipitate. At +2°C to +8°C (lyophilized) it is stable for several years (dry storage), in solution (-20°C) several years or (+2°C to +8°C) several weeks.

Ribonuclease A is an endo-ribonuclease specifically cleaving single stranded RNA at the 3' side of pyrimidine bases (cytosine and uracil). RNAse A ist used to prepare RNA-free DNA, to digest non-hybridised regions of RNA-DNA hybrids and as a molecular weight marker. The pH optimum for RNAse A is between 7.0-7.5. RNAse A is inhibited by Diethylpyrocarbonate (DEPC) >, guanidinium salt (4M GuaSCN), beta-Mercaptoethanol, heavy metals, vanadyl-ribonucleoside complexes, RNAse-inhibitor from human placenta and by competitive DNA. RNAse A cleaves single- and double-stranded RNA and RNA in RNA:DNA hybrids at low salt conditions (100 mM NaCl). At high salt conditions (>300 mM NaCl), RNAse A cleaves single-stranded RNA only. The enzyme can only be removed by Proteinase K digest and subsequent phenol extraction. Elimination of DNAse activity: Product S5231 (RNAse A (not certified DNAse free)) may contain DNAse activity. As RNAse A is heat stable it is recommended to heat inactivate DNAse activity before use. Procedure: 10mg/mL RNAse A in 0.01 sodium acetate (pH5.2) have to be heated to 100°C for 15 minutes in a water bath. After 15 minutes, the RNAse shall remain in the water bath while the water cools down to room temperature. pH can be adjusted by addition of 0.1 time the volume of 1M Tris-HCl (pH7.4). After aliquotation of the now DNAse free RNAse A solution, each aliquote should be stored a -20°C. NOTE: RNAse A precipitates if concentrated solutions are heated to 100°C at neutral pH! Stock solution: Stock solutions are prepared at concentrations from 1 - 10mg/mL in 10mM Tris/HCl, pH7.5; 15mM NaCl or in 10mM Tris/HCl, pH7.5; 1mM EDTA, pH8.0 (TE buffer).The recommended working concentration is 10µg/mL (removal of RNA from plasmid preparations; 1hr, RT) or 100ng/mL (preparation of 'blunt ends' of double-stranded cDNA). Stability:RNase A aggregates during lyophilizing and storage. It has a high affinity to glas surfaces, which has to be taken into consideration. At neutral pH (e. g. in PBS pH 7.4) and high concentrations (>10mg/mL) the enzyme will precipitate. At +2°C to +8°C (lyophilized) it is stable for several years (dry storage), in solution (-20°C) several years or (+2°C to +8°C) several weeks.

Lysozyme (Muramidase) preferentially hydrolyses the β-1,4-glycosidic binding between N-Acetyl muraminic acid and N-Acetyl glucosamine, a component of the proteoglycan-cell wall of certain microorganisms. The enzyme is present in many organisms. In molecular biology, the enzyme from chicken white egg is used to lyse E. coli for the isolation of plasmid-DNA. The working concentration is 200-300 μg/µL. To increase the plasmid yield (approx. 5-10%) lysozyme may be added. Another application is the lysis of bacteria for the preparation of bacterial RNA. In this case the working concentration is 40μg/mL (stock solution 50mg/mL). Stability: The lyophilized powder of lysozyme is stable for many years at +2°C to +8°C. In solution, the stability at pH values from 4 to 5 at +2°C to +8°C is several weeks and at room temperature several days. pH-optimum is at 9.2, the isoelectric point at 11.0. Lysozyme will be inhibited by surfactants like SDS or alcohols and fatty acids, imidazole and indol-derivatives.

The enzyme Lysozyme (Muramidase) affects the cell walls of bacteria. Thereby the extraction efficiency of proteins or nucleic acids is significantly improved. Genaxxon Lysozyme from chicken egg is available as lyophilized powder. Lysozyme preferentially hydrolyses the β-1,4-glycosidic binding between N-Acetyl muraminic acid and N-Acetyl glucosamine, a component of the proteoglycan-cell wall of certain microorganisms. The enzyme is present in many organisms. In molecular biology, the enzyme from chicken white egg is used to lyse E. coli for the isolation of plasmid-DNA. The lysis of E.coli is improved by the combined addition of both lysozyme and and a nuclease such as DNase I (M3028) >. The working concentration is 200-300 μg/µL. To increase the plasmid yield (approx. 5-10%), lysozyme may be added. Another application is the lysis of bacteria for the preparation of bacterial RNA. In this case the working concentration is 40μg/mL (stock solution 50mg/mL). Lysozyme from chicken egg is most effective for the lysis of gram-positive bacteria. Besides this, it facilitates the lysis of gram-negative bacteria such as Salmonella and Shigella. Lyophilized, white powder Enzyme for lysis of bacterial cell walls Improvement of protein purification from inclusion bodies Activity: min. 20.000 U/mg (pH: 6.25) Lysozyme will be inhibited by surfactants like SDS (M3292) > or alcohols and fatty acids, Imidazole (M6033) > and Indol-derivatives. Stability: The lyophilized powder of lysozyme is stable for many years at +2°C to +8°C. In solution, the stability at pH values from 4 to 5 at +2°C to +8°C is several weeks and at room temperature several days. pH-optimum is at 9.2, the isoelectric point at 11.0.

Hyaluronidase isolated from sheep testes. The mammalian glycolytic hyaluronidase (EC 3.2.1.35) catalyzes the hydrolysis of the 1-4 bond between N-acetyl-D-glucosamine and D-glucuronic acid in hyaluronic acid. It also hydrolyses 1,4-beta-D-glycosidic linkages between N-acetyl-galactosamine or N-acetylgalactosamine sulfate and glucuronic acid in chondroitin sulfates A and C, and dermatan. pH-Optimum of the enzyme: 7.0-8.0. Stock solution: 1-300mg/mL in phosphate buffer or water. Working solution: 0.1-10mg/mL. Synonyms: Hyaluronoglucosidase, Hyaluronate 4-glycanohydrolase, Mucinase.

T4 DNA Ligase catalyzes the formation of a phosphodiester bonds between 5' phosphate and 3' hydroxyl termini in duplex DNA/RNA. The enzyme can join blunt end and cohesive end termini, repair single stranded nicks in duplex DNA, RNA, or DNA/RNA hybrids.Cohesive End Ligation:For most cohesive end ligations, a 30 minute incubation at 20°C is sufficient. Incubations at 16°C for 4-16 hours are routinely used for the majority of applications. Ligation of blunt ends and single-base pair overhang fragments requires more enzyme to achieve the same extent of ligation as cohesive end DNA fragments. Ligation may be enhanced by addition of PEG, or by reducing the rATP concentration. T4 DNA Ligase requires ATP > as a cofactor. For PCR we recommend our cost-efficient Taq DNA Polymerase S (M3001 >), or our Pfu Proofreading Polymerase (M3004 >). Applications • Cloning of restriction enzymes generated DNA fragments • Cloning of PCR products • Connection of double-stranded oligonucleotide linkers or adapters with DNA • site-specific mutagenesis • Amplified fragment length polymorphism (AFLP) • Nicking repair in duplex DNA, RNA or DNA/RNA hybrids • Self-circulation of linear DNA.Definition of T4 Ligase activity2 different definitions of T4 Ligase activities are used!One Weiss unit is defined as the amount of enzyme required to convert 1 nmol of 32P-labeled inorganic pyrophosphate into Norit adsorbable material in 20 minutes at 37°C, using specified reaction conditions (Weiss, B., et al., (1968) J. Biol. Chem., 243, 4543). Norit is a type of activated carbon.A Cohesive End Unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of lambda DNA (5´ DNA termini concentration of 0.12µM (300µg/mL)) in 20µL of 1X T4 DNA Ligase Buffer in 30 minutes at 16°C. The conversion factor between the two activities is: One Cohesive End Ligation units (CEL units) corresponds to 0.015 Weiss units. One Weiss unit therefore corresponds to 66.67 Cohesive End Ligation units (CEL units).