Shop All DNA⁄RNA Modifying Enzymes
RapidOut DNA Removal Kit (Thermo Scientific™)
Thermo Scientific RapidOut DNA Removal Kit rapidly and safely removes genomic DNA from total RNA and mRNA preparations. Complete digestion of DNA and safe removal of DNase I from the digestion reaction is ensured without RNA damaging steps, such as heating or organic extraction. First, the RNA sample is treated with recombinant RNase-free DNase I to levels below the limit of detection by routine PCR. DNase I is safely removed subsequently using proprietary DNase Removal Reagent (DRR).
DRR efficiently binds DNase I and the complex is collected at the bottom of the tube by centrifugation. The purified RNA is collected as a supernatant. The RNA after RapidOut procedure is free from DNA contamination and free of DNase I. It is ready to use in different applications including end-point or real-time RT-PCR, cloning, microarrays, and Northern blotting.
Highlights
• Efficient—complete ds and ssDNA digestion and proprietary technology for DNase I removal
• Rapid—single step sufficient for complete DNase I removal
• Safe—no need for toxic organic extractions or RNA-damaging heating steps
Applications
Main Applications
• RNA isolation and RNA analysis, particularly RT-qPCR and RT-PCR (customers performing expression analysis of low transcription level genes.
• Customers performing ds-cDNA synthesis from total RNA preps.
Other applications
• Elimination of DNA from RNA for microinjections and transfection.
• Elimination of DNA from RNA prior microarray analysis.
• Elimination of DNA from RNA prior Northern blot analysis.
DRR efficiently binds DNase I and the complex is collected at the bottom of the tube by centrifugation. The purified RNA is collected as a supernatant. The RNA after RapidOut procedure is free from DNA contamination and free of DNase I. It is ready to use in different applications including end-point or real-time RT-PCR, cloning, microarrays, and Northern blotting.
Highlights
• Efficient—complete ds and ssDNA digestion and proprietary technology for DNase I removal
• Rapid—single step sufficient for complete DNase I removal
• Safe—no need for toxic organic extractions or RNA-damaging heating steps
Applications
Main Applications
• RNA isolation and RNA analysis, particularly RT-qPCR and RT-PCR (customers performing expression analysis of low transcription level genes.
• Customers performing ds-cDNA synthesis from total RNA preps.
Other applications
• Elimination of DNA from RNA for microinjections and transfection.
• Elimination of DNA from RNA prior microarray analysis.
• Elimination of DNA from RNA prior Northern blot analysis.
T4 DNA Ligase (1 U/µL) (Invitrogen™)
La ligase d’ADN T4 catalyse la formation de liaisons phosphodiesters en présence d’ATP entre les ADN bicaténaires avec des extrémités 3´-hydroxyle et 5´-phosphate. L’unique tampon de ligase d’ADN T4 optimise la ligature, ce qui peut être réalisé en 5 minutes. Les acides nucléiques monocaténaires ne sont pas des substrats pour cette enzyme.
Applications
Clonage (ligature des extrémités franches ou cohésives) et ajout de liants ou d’adaptateurs à l’ADN aux extrémités franches
Source
Purifié à partir de l’E. coli lambda lysogène NM989
Performance et tests de qualité
Essais d’endodésoxyribonucléase et d’exodésoxyribonucléase 3´ et 5´ ; testé pour l’efficacité de la ligature
Définition de l’unité
Une unité catalyse l’échange d’1 nmol 32 de pyrophosphate marqué P dans l’ATP en 20 min à 37°C. Une unité est égale à approximativement 300 unités de ligature d’extrémités cohésives.
Conditions de réaction de l’unité
66 mM de Tris-HCl (pH 7,6), 6,6 mM de MgCl2, 10 mM de DTT, 66 µM d’ATP, 3,3 µM 32 de pyrophosphate marqué P et enzyme dans 0,1 ml pendant 20 min à 37°C.
Applications
Clonage (ligature des extrémités franches ou cohésives) et ajout de liants ou d’adaptateurs à l’ADN aux extrémités franches
Source
Purifié à partir de l’E. coli lambda lysogène NM989
Performance et tests de qualité
Essais d’endodésoxyribonucléase et d’exodésoxyribonucléase 3´ et 5´ ; testé pour l’efficacité de la ligature
Définition de l’unité
Une unité catalyse l’échange d’1 nmol 32 de pyrophosphate marqué P dans l’ATP en 20 min à 37°C. Une unité est égale à approximativement 300 unités de ligature d’extrémités cohésives.
Conditions de réaction de l’unité
66 mM de Tris-HCl (pH 7,6), 6,6 mM de MgCl2, 10 mM de DTT, 66 µM d’ATP, 3,3 µM 32 de pyrophosphate marqué P et enzyme dans 0,1 ml pendant 20 min à 37°C.
Shrimp Alkaline Phosphatase (SAP) (Applied Biosystems™)
Proven Performance – the Phosphatase benchmark
•100% heat-inactivated in 5 min at 65°C
•Significantly improved storage stability at lower temperatures (see Fig. 1 and 2)
•Very high specific activity (see Fig. 3)
•Removes 5'-phosphates from DNA, RNA, dNTPs, and proteins
•Purified from a recombinant source
•May be added directly to restriction enzyme digests
•No vector purification necessary
•Requires no supplemental zinc or other additives for activity
•Works direct in many different buffers
•Easy treatment of unincorporated dNTPs in PCR products prior to DNA sequencing or SNP analysis
USB Shrimp Alkaline Phosphatase (SAP)
Shrimp Alkaline Phosphatase (SAP) is a high specific activity, heat-labile alkaline phosphatase purified from a recombinant source and originally isolated from Pandalus borealis (arctic shrimp). SAP is useful in many molecular biology applications such as the dephosphorylation of phosphorylated ends of DNA or RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents relegation of linearized plasmid DNA. SAP may also be used to treat unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.
Shrimp Alkaline Phosphatase has approximately the same specific activity as Calf Intestinal Alkaline Phosphatase (CIAP), and like CIAP, is active in virtually all restriction enzyme reaction buffers. Unlike CIAP, Shrimp Alkaline Phosphatase is completely and irreversibly inactivated by heating reactions at 65°C for 15 min.
Shrimp Alkaline Phosphatase is particularly useful in preparing PCR products for applications involving sequencing, SNP analysis or labeling methods. Typically, excess dNTPs remaining after PCR interfere with subsequent enzymatic reactions involving DNA synthesis. SAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step.
We are pleased to be offering a recombinant version of our phosphatase benchmark. Recombinant SAP eliminates the dependence on animal sourcing and offers the added benefits of increased storage stability and batch to batch consistency while providing exceptional enzymatic activity and 100% heat inactivation.
Properties:
Molecular Weight: Homodimer. Monomer is 55 kDa as determined by amino acid sequence.
Optimum pH: 10.4 in glycine buffer and pH 8.0 in Tris buffer.
Optimum Temperature: 37°C
Heat-Inactivation: 65°C for 15 min.
Inhibitors: 10mM DTT, 0.1% β-ME
Reaction Conditions: Active in NaCl, KCl. Requires Mg2+ for highest activity.
Source:
Recombinant
Purity:
Tested for contaminating endonucleases, exonucleases, and ribonucleases.
Storage Buffer:
25mM Tris-HCl (pH 7.5), 1mM MgCl2, 50% glycerol.
Assay Conditions:
The reaction mixture contains 100mM glycine, pH 10.4, 1mM MgCl2, 1mM ZnCl2, 10mM p-nitrophenyl phosphate, and 0.001-0.1 units of Shrimp Alkaline Phosphatase (SAP). The change in absorbance at 405 nm is monitored (3050 µL reaction volume).
Unit Definition:
One unit is the amount of enzyme which catalyzes the hydrolysis of 1 µmol of p-nitrophenyl phosphate per min in glycine buffer (pH 10.4) at 37°C.
Concentration:
1 unit/µL
Functional Test:
Dephosphorylation of restriction enzyme digested plasmids (5 – 20 pmol of 5'-ends, 0.1 -0.5 units/pmol 5'-ends). Reduces religation to < 0.5% compared to the untreated control.
PROTOCOL FOR DEPHOSPHORYLATION OF NUCLEOTIDES AND DEGRADATION OF PRIMERS PRIOR TO SEQUENCING REACTIONS OR SNP ANALYSES:
Please refer to the USB ExoSAP-IT protocol, the benchmark in PCR clean-up.
The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP.
Functionally Tested 10X SAP Reaction Buffer (Included, PN 70103):
200mM Tris-HCl (pH 8.0), 100mM MgCl2.
Functionally Tested SAP Dilution Buffer (1 ml included, PN 72761):
50mM Tris-HCl (pH 8.0).
References:
1. RUAN, C. C., SAMOLS, S. B. AND FULLER, C. W. (1990) Comments 17, (No.1), United States Biochemical Corporation, Cleveland, OH.
2. WERLE, E., SCNEIDER C., RENNER, M., VÖLKER, M. AND FIEHN, W. (1994) Nucleic Acids Res. 22, 4354-4355.
3. HANKE, M. AND WINK, M. (1994) BioTechniques17, 858-860.
•100% heat-inactivated in 5 min at 65°C
•Significantly improved storage stability at lower temperatures (see Fig. 1 and 2)
•Very high specific activity (see Fig. 3)
•Removes 5'-phosphates from DNA, RNA, dNTPs, and proteins
•Purified from a recombinant source
•May be added directly to restriction enzyme digests
•No vector purification necessary
•Requires no supplemental zinc or other additives for activity
•Works direct in many different buffers
•Easy treatment of unincorporated dNTPs in PCR products prior to DNA sequencing or SNP analysis
USB Shrimp Alkaline Phosphatase (SAP)
Shrimp Alkaline Phosphatase (SAP) is a high specific activity, heat-labile alkaline phosphatase purified from a recombinant source and originally isolated from Pandalus borealis (arctic shrimp). SAP is useful in many molecular biology applications such as the dephosphorylation of phosphorylated ends of DNA or RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents relegation of linearized plasmid DNA. SAP may also be used to treat unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.
Shrimp Alkaline Phosphatase has approximately the same specific activity as Calf Intestinal Alkaline Phosphatase (CIAP), and like CIAP, is active in virtually all restriction enzyme reaction buffers. Unlike CIAP, Shrimp Alkaline Phosphatase is completely and irreversibly inactivated by heating reactions at 65°C for 15 min.
Shrimp Alkaline Phosphatase is particularly useful in preparing PCR products for applications involving sequencing, SNP analysis or labeling methods. Typically, excess dNTPs remaining after PCR interfere with subsequent enzymatic reactions involving DNA synthesis. SAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step.
We are pleased to be offering a recombinant version of our phosphatase benchmark. Recombinant SAP eliminates the dependence on animal sourcing and offers the added benefits of increased storage stability and batch to batch consistency while providing exceptional enzymatic activity and 100% heat inactivation.
Properties:
Molecular Weight: Homodimer. Monomer is 55 kDa as determined by amino acid sequence.
Optimum pH: 10.4 in glycine buffer and pH 8.0 in Tris buffer.
Optimum Temperature: 37°C
Heat-Inactivation: 65°C for 15 min.
Inhibitors: 10mM DTT, 0.1% β-ME
Reaction Conditions: Active in NaCl, KCl. Requires Mg2+ for highest activity.
Source:
Recombinant
Purity:
Tested for contaminating endonucleases, exonucleases, and ribonucleases.
Storage Buffer:
25mM Tris-HCl (pH 7.5), 1mM MgCl2, 50% glycerol.
Assay Conditions:
The reaction mixture contains 100mM glycine, pH 10.4, 1mM MgCl2, 1mM ZnCl2, 10mM p-nitrophenyl phosphate, and 0.001-0.1 units of Shrimp Alkaline Phosphatase (SAP). The change in absorbance at 405 nm is monitored (3050 µL reaction volume).
Unit Definition:
One unit is the amount of enzyme which catalyzes the hydrolysis of 1 µmol of p-nitrophenyl phosphate per min in glycine buffer (pH 10.4) at 37°C.
Concentration:
1 unit/µL
Functional Test:
Dephosphorylation of restriction enzyme digested plasmids (5 – 20 pmol of 5'-ends, 0.1 -0.5 units/pmol 5'-ends). Reduces religation to < 0.5% compared to the untreated control.
PROTOCOL FOR DEPHOSPHORYLATION OF NUCLEOTIDES AND DEGRADATION OF PRIMERS PRIOR TO SEQUENCING REACTIONS OR SNP ANALYSES:
Please refer to the USB ExoSAP-IT protocol, the benchmark in PCR clean-up.
The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP.
Functionally Tested 10X SAP Reaction Buffer (Included, PN 70103):
200mM Tris-HCl (pH 8.0), 100mM MgCl2.
Functionally Tested SAP Dilution Buffer (1 ml included, PN 72761):
50mM Tris-HCl (pH 8.0).
References:
1. RUAN, C. C., SAMOLS, S. B. AND FULLER, C. W. (1990) Comments 17, (No.1), United States Biochemical Corporation, Cleveland, OH.
2. WERLE, E., SCNEIDER C., RENNER, M., VÖLKER, M. AND FIEHN, W. (1994) Nucleic Acids Res. 22, 4354-4355.
3. HANKE, M. AND WINK, M. (1994) BioTechniques17, 858-860.
Ambion™ T4 RNA Ligase, cloned, 5 U/µL (Invitrogen™)
Ambion® T4 RNA Ligase catalyzes the formation of a phosphodiester linkage between a 5'-phosphoryl-terminated nucleic acid donor and a 3'-hydroxyl-terminated nucleic acid acceptor. Substrates include RNA, DNA, and oligonucleotides. One tube containing 2,500 U (5 U/ µL) is provided along with 10X Reaction Buffer. The enzyme can be used for tagging the 5' ends of mRNA with oligonucleotides for mapping studies (RACE), 3'-end labeling RNA molecules, and circularizing RNA and DNA molecules.
Unit Definition:
One unit catalyzes the formation of 1 nmol of [5'-32P]-rA12-18 into a phosphatase-resistant form in 30 min at 37°C.
Unit Definition:
One unit catalyzes the formation of 1 nmol of [5'-32P]-rA12-18 into a phosphatase-resistant form in 30 min at 37°C.
Rapid DNA Ligation Kit (Thermo Scientific™)
Thermo Scientific Rapid DNA Ligation Kit enables fast sticky-end or blunt-end DNA ligation in only 5 minutes at room temperature.The kit contains T4 DNA ligase and a specially-formulated 5X rapid ligation buffer optimized for fast and efficient DNA ligation. Fast ligation efficiency is equal to that obtained with T4 DNA ligase in a standard 1 hour ligation. The ligation reaction mixture can be used directly for bacterial transformation with the TransformAid Bacterial Transformation Kit or with other conventional transformation procedures.
Highlights
• Fast—ligation of either sticky- or blunt-end DNA in only 5 minutes
• Convenient—reaction mixture can be used directly for bacterial transformation
Applications
• Routine cloning experiments
• Blunt-end cloning
• Library construction
• TA cloning
Includes
• T4 DNA Ligase
• 5X Rapid Ligation Buffer
• Water, nuclease-free
• Detailed Protocol
Note
Prior to electroporation, it is necessary to inactivate T4 DNA ligase by chloroform extraction or spin column purification, e.g. with Thermo Scientific GeneJET PCR Purification Kit (K0701).
Related Products
Rapid DNA Ligation Kit
Highlights
• Fast—ligation of either sticky- or blunt-end DNA in only 5 minutes
• Convenient—reaction mixture can be used directly for bacterial transformation
Applications
• Routine cloning experiments
• Blunt-end cloning
• Library construction
• TA cloning
Includes
• T4 DNA Ligase
• 5X Rapid Ligation Buffer
• Water, nuclease-free
• Detailed Protocol
Note
Prior to electroporation, it is necessary to inactivate T4 DNA ligase by chloroform extraction or spin column purification, e.g. with Thermo Scientific GeneJET PCR Purification Kit (K0701).
Related Products
Rapid DNA Ligation Kit
Exonuclease I, standard concentration, (10 units/µL) (Applied Biosystems™)
Description:
Exonuclease I hydrolyzes single-stranded DNA in the 3'→5' direction, releasing
5'-mononucleotides and leaving the terminal 5'-dinucleotide intact. Hydrolysis is processive and cannot proceed if the 3' terminus is phosphorylated. Exonuclease I can be used to measure the endonucleolytic cleavage of covalently closed circular single-stranded DNA reacted with an endonuclease of interest. In addition, DNA helicase activity can be measured utilizing Exonuclease I.
Exonuclease I is particularly useful in preparing the products of PCR for applications involving sequencing or labeling methods. Typically, the excess primers and any other extraneous single-stranded DNA present in PCR products will interfere with subsequent enzymatic reactions involving DNA synthesis. The hydrolytic properties of Exonuclease I degrade all single-stranded DNA present in the PCR mixture allowing the product to be used more efficiently in other applications. When combined with Shrimp Alkaline Phosphatase (PN 78390) for dNTP dephosphorylation, the use of alternative purification methods, such as columns, gels or magnetic separations, are completely eliminated.
For PCR clean up with Exonuclease I, see the USB ExoSAP-IT protocol. The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP.
Properties:
Molecular Weight: 55 kDa
Heat Inactivation: 80°C for 15 min.
Degrades to terminal dinucleotides.
Degrades glycosylated DNA.
Optimum Temperature: 37°C
Purity:
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating endonucleases, double-stranded exonucleases, and ribonucleases.
Storage Buffer:
20mM Tris-HCI (pH 7.5), 5mM 2-mercaptoethanol, 0.5mM EDTA, 50% glycerol.
Assay Conditions:
The reaction mixture (100 µL) contains 67mM glycine buffer (pH 9.5), 10 mM
2-mercaptoethanol, 6.7 mM MgCl2, 0.5 mM denatured DNA, and enzyme. Incubation is at 37°C for 30 min.
Unit Definition:
One unit is the amount of enzyme which catalyzes the release of 10 nmol of acid-soluble nucleotide from denatured DNA in 30 min at 37°C under standard conditions.
Concentration:
Standard Conc.: 10 units/µL, PN 70073
High Conc.: 20 units/µL, PN 72073
Functional Assay: Treated PCR product with Exonuclease I to degrade unincorporated primers before performing sequencing reaction with Sequenase™ Version 2.0 DNA Polymerase Sequencing Kit (PN 70170).
References:
GOLDMARK, P. J. AND LINN, S. (1972) J. Biol. Chem. 247, 1849-1860.
ROSAMOND, J., TELANDER, K. M. AND LINN, S. (1979) J. Biol. Chem. 254, 8646-8652.
WERLE, E., SCNEIDER C., RENNER, M., VÖLKER, M. AND FIEHN, W. (1994) Nucleic Acids Res. 22, 4354-4355.
HANKE, M. AND WINK, M. (1994) BioTechniques 17, 858-860.
Exonuclease I hydrolyzes single-stranded DNA in the 3'→5' direction, releasing
5'-mononucleotides and leaving the terminal 5'-dinucleotide intact. Hydrolysis is processive and cannot proceed if the 3' terminus is phosphorylated. Exonuclease I can be used to measure the endonucleolytic cleavage of covalently closed circular single-stranded DNA reacted with an endonuclease of interest. In addition, DNA helicase activity can be measured utilizing Exonuclease I.
Exonuclease I is particularly useful in preparing the products of PCR for applications involving sequencing or labeling methods. Typically, the excess primers and any other extraneous single-stranded DNA present in PCR products will interfere with subsequent enzymatic reactions involving DNA synthesis. The hydrolytic properties of Exonuclease I degrade all single-stranded DNA present in the PCR mixture allowing the product to be used more efficiently in other applications. When combined with Shrimp Alkaline Phosphatase (PN 78390) for dNTP dephosphorylation, the use of alternative purification methods, such as columns, gels or magnetic separations, are completely eliminated.
For PCR clean up with Exonuclease I, see the USB ExoSAP-IT protocol. The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP.
Properties:
Molecular Weight: 55 kDa
Heat Inactivation: 80°C for 15 min.
Degrades to terminal dinucleotides.
Degrades glycosylated DNA.
Optimum Temperature: 37°C
Purity:
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating endonucleases, double-stranded exonucleases, and ribonucleases.
Storage Buffer:
20mM Tris-HCI (pH 7.5), 5mM 2-mercaptoethanol, 0.5mM EDTA, 50% glycerol.
Assay Conditions:
The reaction mixture (100 µL) contains 67mM glycine buffer (pH 9.5), 10 mM
2-mercaptoethanol, 6.7 mM MgCl2, 0.5 mM denatured DNA, and enzyme. Incubation is at 37°C for 30 min.
Unit Definition:
One unit is the amount of enzyme which catalyzes the release of 10 nmol of acid-soluble nucleotide from denatured DNA in 30 min at 37°C under standard conditions.
Concentration:
Standard Conc.: 10 units/µL, PN 70073
High Conc.: 20 units/µL, PN 72073
Functional Assay: Treated PCR product with Exonuclease I to degrade unincorporated primers before performing sequencing reaction with Sequenase™ Version 2.0 DNA Polymerase Sequencing Kit (PN 70170).
References:
GOLDMARK, P. J. AND LINN, S. (1972) J. Biol. Chem. 247, 1849-1860.
ROSAMOND, J., TELANDER, K. M. AND LINN, S. (1979) J. Biol. Chem. 254, 8646-8652.
WERLE, E., SCNEIDER C., RENNER, M., VÖLKER, M. AND FIEHN, W. (1994) Nucleic Acids Res. 22, 4354-4355.
HANKE, M. AND WINK, M. (1994) BioTechniques 17, 858-860.
Ribonuclease H, from E. coli (cloned) 10 U/μL (Invitrogen™)
Ambion® Ribonuclease H (RNase H) is isolated from an E. coli strain that over-expresses the gene. RNase H specifically degrades the RNA in RNA:DNA hybrids to produce 3' -hydroxyl and 5' -phosphate terminated products. It is supplied in one tube containing 200 U (10 U/ µL). The enzyme will not degrade DNA or unhybridized RNA. RNase H is an integral part of most RNA amplification and NASBA protocols. It can also be used to degrade specific RNAs when the complementary DNA oligo is hybridized, such as poly(A) tail removal from mRNA hybridized to oligo(dT). Ribonuclease H is rigorously tested for contaminating nonspecific endonuclease, exonuclease, RNase, and protease activity.
Unit Definition:
One unit of Ribonuclease H is the amount of enzyme required to increase fluorescence 1.5 RFUs per sec at 37°C using 20 pmol of RNaseAlert® probe coupled to 1,000 pmol of a complementary oligonucleotide as substrate.
Unit Definition:
One unit of Ribonuclease H is the amount of enzyme required to increase fluorescence 1.5 RFUs per sec at 37°C using 20 pmol of RNaseAlert® probe coupled to 1,000 pmol of a complementary oligonucleotide as substrate.
MuA Transposase (Thermo Scientific™)
Thermo Scientific transposon products are based on the transposition machinery of the bacteriophage Mu. During the lytic phase of the phage's life cycle the machinery replicates its genome by transposing repeatedly inside the host genome. The Mu transposition reaction has been modified into an in vitro reaction catalyzed by a single enzyme - MuA Transposase. In this system, one in vitro reaction is capable of generating more than a million transposon insertion clones.
The Mutation Generation System (MGS Kit) and Stop Generation System (STOP Kit) were developed for functional analysis of proteins. These new transposon tools enable the creation of saturated libraries of mutated proteins in a single reaction with less hands-on time than any other method. The location of the transposon insertion in each mutated clone can be mapped by either PCR or sequencing. With MGS and STOP kits, thousands of mutated clones are ready for expression studies in just 2 to 3 days.
The MGS Kit contains the complete set of reagents for transposon-based linker scanning mutagenesis of any target protein. The MGS Entranceposons are designed for making subtle changes in the structure of a target protein by inserting 15 bp in-frame linkers throughout the corresponding target gene. This in-frame insertion allows for conservation of downstream sequences.
The STOPKit Entranceposons contain translational stop codons in all three reading frames within the terminal portion of the transposon sequence. The proprietary modification of the Stop Generation System makes it possible to generate a saturated C-terminal deletion library from virtually any target protein with a maximum addition of three amino acids.
Features:
• Efficient—Create saturated insertion libraries for sequencing and protein analysis in a single reaction
• Fast—Decrease hands-on time compared to conventional methods
• Random—Eliminate target site preference or insertion hot-spot
Applications
The STOP Kit generates truncated proteins for functional assays of:
• Enzymes
• Receptors
• Structural proteins etc.
The MGS Kit generates random fifteen basepair in vitro insertions into any target DNA for:
• Rapid generation of in-frame five amino acid insertion libraries of any protein for functional analyses
• Rapid and random mutagenesis of cloned promoters and other regulatory DNA regions
• Random insertion of a NotI restriction enzyme site into any target DNA clone
Advantages
MGS Kit
• Thousands of different insertion clones from a single reaction
• Generates random insertions of 5 amino acids in all 3 reading frames
• Short in-frame insertions; no stop codons
• Flexibility in mapping mutants of interest: mutations are easily mapped by NotI or PCR
• Faster and more effective than linker scanning mutagenesis
• STOP Kit
• Saturated library of truncated proteins from a single reaction in two days
• Translational STOP codon in all three reading frames
• The target DNA sequence can be unknown
• Faster and more effective than conventional methods
• No specific primers required
Related Products
MuA Transposase (concentrated)
Mutation Generation System Kit
The Mutation Generation System (MGS Kit) and Stop Generation System (STOP Kit) were developed for functional analysis of proteins. These new transposon tools enable the creation of saturated libraries of mutated proteins in a single reaction with less hands-on time than any other method. The location of the transposon insertion in each mutated clone can be mapped by either PCR or sequencing. With MGS and STOP kits, thousands of mutated clones are ready for expression studies in just 2 to 3 days.
The MGS Kit contains the complete set of reagents for transposon-based linker scanning mutagenesis of any target protein. The MGS Entranceposons are designed for making subtle changes in the structure of a target protein by inserting 15 bp in-frame linkers throughout the corresponding target gene. This in-frame insertion allows for conservation of downstream sequences.
The STOPKit Entranceposons contain translational stop codons in all three reading frames within the terminal portion of the transposon sequence. The proprietary modification of the Stop Generation System makes it possible to generate a saturated C-terminal deletion library from virtually any target protein with a maximum addition of three amino acids.
Features:
• Efficient—Create saturated insertion libraries for sequencing and protein analysis in a single reaction
• Fast—Decrease hands-on time compared to conventional methods
• Random—Eliminate target site preference or insertion hot-spot
Applications
The STOP Kit generates truncated proteins for functional assays of:
• Enzymes
• Receptors
• Structural proteins etc.
The MGS Kit generates random fifteen basepair in vitro insertions into any target DNA for:
• Rapid generation of in-frame five amino acid insertion libraries of any protein for functional analyses
• Rapid and random mutagenesis of cloned promoters and other regulatory DNA regions
• Random insertion of a NotI restriction enzyme site into any target DNA clone
Advantages
MGS Kit
• Thousands of different insertion clones from a single reaction
• Generates random insertions of 5 amino acids in all 3 reading frames
• Short in-frame insertions; no stop codons
• Flexibility in mapping mutants of interest: mutations are easily mapped by NotI or PCR
• Faster and more effective than linker scanning mutagenesis
• STOP Kit
• Saturated library of truncated proteins from a single reaction in two days
• Translational STOP codon in all three reading frames
• The target DNA sequence can be unknown
• Faster and more effective than conventional methods
• No specific primers required
Related Products
MuA Transposase (concentrated)
Mutation Generation System Kit
DNase I Solution (2500 U/mL) (Thermo Scientific™)
Thermo Scientific DNase I removes unwanted DNA from cell lysates to improve protein extraction efficiency.
Features of Thermo Scientific DNase I:
• Degrades and removes unwanted DNA from samples
• Cleaves both single-stranded and double-stranded DNA
• Compatible with Thermo Scientific Pierce Cell Lysis Reagents
• Reduces viscosity of bacterial lysates (protein extracts) to facilitate pipetting
Deoxyribonuclease I (DNase I) is a single, glycosylated polypeptide that degrades unwanted single- and double-stranded DNA. The enzyme works by cleaving DNA into 5' phosphodinucleotide and small oligonucleotide fragments. DNase I is commonly added to cell lysis reagents to remove the viscosity caused by the DNA content in bacterial cell lysates or to remove the DNA templates from RNAs produced by in vitro transcription. This grade of DNase is sufficient for protein work. Use RNase-free DNase for any application requiring the digestion of DNA in which it is crucial to avoid damage to RNA.
General information about the use of DNase I:
• Calcium ions are required for activity of DNase I. Trace amounts of Ca++ may be present at high enough concentration for DNase I to be active, however use of EGTA or calcium-free buffers can reduce DNase I activity to undetectable levels.
• High levels (i.e., 100 mM) of monovalent ions such as Na+ and K+ will decrease DNase I activity
• DNase I is inactivated by heating to 65°C for 10 minutes
• Kunitz unit: 1 Kunitz unit is the amount of enzyme required to cause an increase of 0.001 A260nm/min/mL at 25°C in 0.1M NaOAc, pH 5.0 due to degradation of highly polymerized DNA
• Degradation assay units: 1 unit is defined as the amount of enzyme required to completely degrade 1 µg of plasmid DNA in the 10 minutes at 37°C in 10 mM Tris·HCl, pH 7.5, 50 mM MgCl2, 13 mM CaCl2.
Specifications:
• Quantity: 0.5 mL
• Concentration: ≥ 2500 units/mL
• Unit definition: 1 unit is defined as the amount of enzyme required to produce an increase in absorbance at 260nm of 0.001/min/mL at 25°C of highly polymerized DNA.
• Visual: Clear, colorless liquid, free of insoluble material
• Formulation: DNase I in 10 mM Tris-HCl pH 7.5, 10 mM CaCl2, 10 mM MgCl2
Related Products
DNase I Solution (1 unit/µL), RNase-free
Features of Thermo Scientific DNase I:
• Degrades and removes unwanted DNA from samples
• Cleaves both single-stranded and double-stranded DNA
• Compatible with Thermo Scientific Pierce Cell Lysis Reagents
• Reduces viscosity of bacterial lysates (protein extracts) to facilitate pipetting
Deoxyribonuclease I (DNase I) is a single, glycosylated polypeptide that degrades unwanted single- and double-stranded DNA. The enzyme works by cleaving DNA into 5' phosphodinucleotide and small oligonucleotide fragments. DNase I is commonly added to cell lysis reagents to remove the viscosity caused by the DNA content in bacterial cell lysates or to remove the DNA templates from RNAs produced by in vitro transcription. This grade of DNase is sufficient for protein work. Use RNase-free DNase for any application requiring the digestion of DNA in which it is crucial to avoid damage to RNA.
General information about the use of DNase I:
• Calcium ions are required for activity of DNase I. Trace amounts of Ca++ may be present at high enough concentration for DNase I to be active, however use of EGTA or calcium-free buffers can reduce DNase I activity to undetectable levels.
• High levels (i.e., 100 mM) of monovalent ions such as Na+ and K+ will decrease DNase I activity
• DNase I is inactivated by heating to 65°C for 10 minutes
• Kunitz unit: 1 Kunitz unit is the amount of enzyme required to cause an increase of 0.001 A260nm/min/mL at 25°C in 0.1M NaOAc, pH 5.0 due to degradation of highly polymerized DNA
• Degradation assay units: 1 unit is defined as the amount of enzyme required to completely degrade 1 µg of plasmid DNA in the 10 minutes at 37°C in 10 mM Tris·HCl, pH 7.5, 50 mM MgCl2, 13 mM CaCl2.
Specifications:
• Quantity: 0.5 mL
• Concentration: ≥ 2500 units/mL
• Unit definition: 1 unit is defined as the amount of enzyme required to produce an increase in absorbance at 260nm of 0.001/min/mL at 25°C of highly polymerized DNA.
• Visual: Clear, colorless liquid, free of insoluble material
• Formulation: DNase I in 10 mM Tris-HCl pH 7.5, 10 mM CaCl2, 10 mM MgCl2
Related Products
DNase I Solution (1 unit/µL), RNase-free
T4 DNA Ligase (5 U/µL) (Invitrogen™)
La ligase d’ADN T4 catalyse la formation de liaisons phosphodiesters en présence d’ATP entre les ADN bicaténaires avec des extrémités 3´-hydroxyle et 5´-phosphate. L’unique tampon de ligase d’ADN T4 optimise la ligature, ce qui peut être réalisé en 5 minutes (1). Les acides nucléiques monocaténaires ne sont pas des substrats pour cette enzyme. Un bulletin technique sur la ligase d’ADN T4 est disponible.
Applications : Clonage (ligature d’extrémités franches ou cohésives) (2). Ajout de lieurs ou d’adaptateurs à l’ADN à extrémités franches (2).
Source : Purifié à partir de l’E. coli œ lysogène NM989
Tests de performance et de qualité : Endodésoxyribonucléase, dosages 3´ et 5´ exodésoxyribonucléase ; efficacité de la ligature testée.
Définition de l’unité : Une unité catalyse l’échange de 1 nmole 32 de pyrophosphate marqué P dans l’ATP en 20 min à 37°C. (Une unité est égale à approximativement 300 unités de ligature d’extrémités cohésives).
Conditions de réaction de l’unité : 66 mM de Tris-HCl (pH 7.6), 6,6 mM de MgCl2, 10 mM de DTT, 66 µM d’ATP, 3,3 µM 32 de pyrophosphate marqué P et enzyme dans 0,1 ml pendant 20 min à 37°C.
Applications : Clonage (ligature d’extrémités franches ou cohésives) (2). Ajout de lieurs ou d’adaptateurs à l’ADN à extrémités franches (2).
Source : Purifié à partir de l’E. coli œ lysogène NM989
Tests de performance et de qualité : Endodésoxyribonucléase, dosages 3´ et 5´ exodésoxyribonucléase ; efficacité de la ligature testée.
Définition de l’unité : Une unité catalyse l’échange de 1 nmole 32 de pyrophosphate marqué P dans l’ATP en 20 min à 37°C. (Une unité est égale à approximativement 300 unités de ligature d’extrémités cohésives).
Conditions de réaction de l’unité : 66 mM de Tris-HCl (pH 7.6), 6,6 mM de MgCl2, 10 mM de DTT, 66 µM d’ATP, 3,3 µM 32 de pyrophosphate marqué P et enzyme dans 0,1 ml pendant 20 min à 37°C.
Ambion™ DNase I (RNase-free) (Invitrogen™)
Ambion DNase I (RNase-free) (E.C. 3.1.21.1) is a nonspecific endonuclease that degrades double- and single-stranded DNA and chromatin. It functions by hydrolyzing phosphodiester linkages, producing mono and oligonucleotides with a 5'-phosphate and a 3'-hydroxyl group. RNase-free DNase I is of the highest purity available and is recommended to degrade DNA in the presence of RNA when the absence of RNase is critical to maintain the integrity of the RNA. For example, DNase I is frequently used to remove template DNA following in vitro transcription, and to remove contaminating DNA in total RNA preparations (especially those from transfected cells that may contain plasmid DNA), used for ribonuclease protection assays, cDNA library contraction, and RT-PCR. DNase I requires bivalent cations (Mg2+ and Ca2+ at approximately 5 mM and 0.5 mM, respectively) for maximal activity, and has a pH optimum of 7.8.
RNase-free DNase I outperforms the competition
A research report in BioTechniques (Matthews et al., 32: 1412-1417, 2002) compared RNase contamination in DNase I preparations from Sigma, Roche, Applied Science, Qiagen, and Ambion. The results revealed that "...with the exception of Ambion®'s RNase-free DNase I, the integrity of cRNA from in vitro transcription reactions was compromised and was still contaminated with DNA. Ambion®'s DNase was used for the remaining experiments requiring DNase digestion...". Ambion® DNase I is tested for contaminating RNase and protease activity. Functionality is determined by digestion of human genomic DNA followed by quantitative real-time PCR to detect undigested DNA.
Unit definition
One unit is the amount of enzyme required to completely degrade 1 µg DNA in 10 min at 37°C, and is equivalent to 0.04 Kunitz units.
Accessory products
For an alternative to bovine DNase I, please consider Recombinant DNase I (Cat. No. AM2235). For a more-active, salt-tolerant DNase, please see the TURBO™ DNase products (Cat. Nos. AM2239 and AM2238).
RNase-free DNase I outperforms the competition
A research report in BioTechniques (Matthews et al., 32: 1412-1417, 2002) compared RNase contamination in DNase I preparations from Sigma, Roche, Applied Science, Qiagen, and Ambion. The results revealed that "...with the exception of Ambion®'s RNase-free DNase I, the integrity of cRNA from in vitro transcription reactions was compromised and was still contaminated with DNA. Ambion®'s DNase was used for the remaining experiments requiring DNase digestion...". Ambion® DNase I is tested for contaminating RNase and protease activity. Functionality is determined by digestion of human genomic DNA followed by quantitative real-time PCR to detect undigested DNA.
Unit definition
One unit is the amount of enzyme required to completely degrade 1 µg DNA in 10 min at 37°C, and is equivalent to 0.04 Kunitz units.
Accessory products
For an alternative to bovine DNase I, please consider Recombinant DNase I (Cat. No. AM2235). For a more-active, salt-tolerant DNase, please see the TURBO™ DNase products (Cat. Nos. AM2239 and AM2238).
Ambion™ RNase A, affinity purified, 1 mg/mL (Invitrogen™)
Ambion® RNase A is an endonuclease that specifically cleaves 3' of U and C residues. Affinity Purified RNase A is intended for critical applications when the absence of DNase and other nonspecific nuclease activities is essential. Supplied in one tube containing 200 µg (1 mg/mL). Note: This preparation contains RNase B, a carbohydrate isoform of RNase A. RNase A is rigorously tested for contaminating nonspecific endonuclease, exonuclease, and protease activity.
T4 beta-glucosyltransferase (Thermo Scientific™)
Thermo Scientific T4 β-glucosyltransferase (T4 BGT) transfers the glucose moiety of uridine diphosphoglucose (UDP-glucose) to the 5-hydroxymethylcytosine (5-hmC) residues in double-stranded DNA generating ß-glucosyl-5 hydroxymethylcytosine.
Thermo Scientific T4 BGT is specifically formulated for fast reaction times without compromising the reaction efficiency. The enzyme completes 5-hmC glucosylation of 1 µg DNA at 37°C in 15 minutes.
Highlights
• Specific—selectively transfers glucose to the hydroxymethyl moiety of 5-hmC
• Fast—complete glucosylation of 1 µg DNA in just 15 min
• Convenient—supplied with optimized buffer and UDP-glucose.
Applications
• Locus specific detection of 5-hmC
• Enrichment of 5-hmC containing DNA
• Labeling of 5-hmC residues using a radioactive UDP-glucose donor
Includes
• T4 β-glucosyltransferase, 5 U/µL
• 10X Epi buffer
• 10X UDP-glucose
Thermo Scientific T4 BGT is specifically formulated for fast reaction times without compromising the reaction efficiency. The enzyme completes 5-hmC glucosylation of 1 µg DNA at 37°C in 15 minutes.
Highlights
• Specific—selectively transfers glucose to the hydroxymethyl moiety of 5-hmC
• Fast—complete glucosylation of 1 µg DNA in just 15 min
• Convenient—supplied with optimized buffer and UDP-glucose.
Applications
• Locus specific detection of 5-hmC
• Enrichment of 5-hmC containing DNA
• Labeling of 5-hmC residues using a radioactive UDP-glucose donor
Includes
• T4 β-glucosyltransferase, 5 U/µL
• 10X Epi buffer
• 10X UDP-glucose
E. coli DNA Ligase (Invitrogen™)
E. coli DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of β-NAD between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate cohesive termini. Single-stranded nucleic acids are not substrates for this enzyme.
Applications: Second-strand cDNA synthesis (1). T4 DNA Ligase alternative when blunt-end ligation is not required (2).
Source: Purified from E. coli 594 (Su- ) bearing λ lysogen gt4lop-11 lig+ S7 (3).
Performance and Quality Testing: Endodeoxyribonuclease and 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency tested.
Unit Definition: One unit is the amount of enzyme required to give 50% ligation of Hind III-digested λ DNA in 30 min. at 16°C in a final volume of 20 µl containing a 5´ termini concentration of 0.12 ´M (300 µg/ml).
Unit Reaction Conditions: 18.8 mM Tris-HCl (pH 8.3), 90.6 mM KCl, 4.6 mM MgCl2 , 3.8 mM DTT, 0.15 mM λ-NAD, 10 mM (NH4 )2 SO4 in 20 µl for 1 h at 16°C.
Applications: Second-strand cDNA synthesis (1). T4 DNA Ligase alternative when blunt-end ligation is not required (2).
Source: Purified from E. coli 594 (Su- ) bearing λ lysogen gt4lop-11 lig+ S7 (3).
Performance and Quality Testing: Endodeoxyribonuclease and 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency tested.
Unit Definition: One unit is the amount of enzyme required to give 50% ligation of Hind III-digested λ DNA in 30 min. at 16°C in a final volume of 20 µl containing a 5´ termini concentration of 0.12 ´M (300 µg/ml).
Unit Reaction Conditions: 18.8 mM Tris-HCl (pH 8.3), 90.6 mM KCl, 4.6 mM MgCl2 , 3.8 mM DTT, 0.15 mM λ-NAD, 10 mM (NH4 )2 SO4 in 20 µl for 1 h at 16°C.
MuA Transposase (concentrated) (Thermo Scientific™)
Thermo Scientific transposon products are based on the transposition machinery of the bacteriophage Mu. During the lytic phase of the phage's life cycle the machinery replicates its genome by transposing repeatedly inside the host genome. The Mu transposition reaction has been modified into an in vitro reaction catalyzed by a single enzyme - MuA Transposase. In this system, one in vitro reaction is capable of generating more than a million transposon insertion clones.
Features
The Mutation Generation System (MGS Kit) and Stop Generation System (STOP Kit) were developed for functional analysis of proteins. These new transposon tools enable the creation of saturated libraries of mutated proteins in a single reaction with less hands-on time than any other method. The location of the transposon insertion in each mutated clone can be mapped by either PCR or sequencing. With MGS and STOP kits, thousands of mutated clones are ready for expression studies in just 2 to 3 days.
The MGS Kit contains the complete set of reagents for transposon-based linker scanning mutagenesis of any target protein. The MGS Entranceposons are designed for making subtle changes in the structure of a target protein by inserting 15 bp in-frame linkers throughout the corresponding target gene. This in-frame insertion allows for conservation of downstream sequences.
The STOPKit Entranceposons contain translational stop codons in all three reading frames within the terminal portion of the transposon sequence. The proprietary modification of the Stop Generation System makes it possible to generate a saturated C-terminal deletion library from virtually any target protein with a maximum addition of three amino acids.
Features:
Efficient—Create saturated insertion libraries for sequencing and protein analysis in a single reaction
Fast—Decrease hands-on time compared to conventional methods
Random—Eliminate target site preference or insertion hot-spot
Applications
The STOP Kit generates truncated proteins for functional assays of:
• Enzymes
• Receptors
• Structural proteins etc.
The MGS Kit generates random fifteen basepair in vitro insertions into any target DNA for:
• Rapid generation of in-frame five amino acid insertion libraries of any protein for functional analyses
• Rapid and random mutagenesis of cloned promoters and other regulatory DNA regions
• Random insertion of a NotI restriction enzyme site into any target DNA clone
Advantages
MGS Kit
• Thousands of different insertion clones from a single reaction
• Generates random insertions of 5 amino acids in all 3 reading frames
• Short in-frame insertions; no stop codons
• Flexibility in mapping mutants of interest: mutations are easily mapped by NotI or PCR
• Faster and more effective than linker scanning mutagenesis
• STOP Kit
• Saturated library of truncated proteins from a single reaction in two days
• Translational STOP codon in all three reading frames
• The target DNA sequence can be unknown
• Faster and more effective than conventional methods
• No specific primers required
Related Products
Mutation Generation System Kit
MuA Transposase
Features
The Mutation Generation System (MGS Kit) and Stop Generation System (STOP Kit) were developed for functional analysis of proteins. These new transposon tools enable the creation of saturated libraries of mutated proteins in a single reaction with less hands-on time than any other method. The location of the transposon insertion in each mutated clone can be mapped by either PCR or sequencing. With MGS and STOP kits, thousands of mutated clones are ready for expression studies in just 2 to 3 days.
The MGS Kit contains the complete set of reagents for transposon-based linker scanning mutagenesis of any target protein. The MGS Entranceposons are designed for making subtle changes in the structure of a target protein by inserting 15 bp in-frame linkers throughout the corresponding target gene. This in-frame insertion allows for conservation of downstream sequences.
The STOPKit Entranceposons contain translational stop codons in all three reading frames within the terminal portion of the transposon sequence. The proprietary modification of the Stop Generation System makes it possible to generate a saturated C-terminal deletion library from virtually any target protein with a maximum addition of three amino acids.
Features:
Efficient—Create saturated insertion libraries for sequencing and protein analysis in a single reaction
Fast—Decrease hands-on time compared to conventional methods
Random—Eliminate target site preference or insertion hot-spot
Applications
The STOP Kit generates truncated proteins for functional assays of:
• Enzymes
• Receptors
• Structural proteins etc.
The MGS Kit generates random fifteen basepair in vitro insertions into any target DNA for:
• Rapid generation of in-frame five amino acid insertion libraries of any protein for functional analyses
• Rapid and random mutagenesis of cloned promoters and other regulatory DNA regions
• Random insertion of a NotI restriction enzyme site into any target DNA clone
Advantages
MGS Kit
• Thousands of different insertion clones from a single reaction
• Generates random insertions of 5 amino acids in all 3 reading frames
• Short in-frame insertions; no stop codons
• Flexibility in mapping mutants of interest: mutations are easily mapped by NotI or PCR
• Faster and more effective than linker scanning mutagenesis
• STOP Kit
• Saturated library of truncated proteins from a single reaction in two days
• Translational STOP codon in all three reading frames
• The target DNA sequence can be unknown
• Faster and more effective than conventional methods
• No specific primers required
Related Products
Mutation Generation System Kit
MuA Transposase
