Shop All DNA⁄RNA Modifying Enzymes

CIAP (Calf Intestinal Alkaline Phosphatase), 20 U/µL (Invitrogen™)

Calf Intestinal Alkaline Phosphatase (CIAP) is a phosphomonoesterase that removes 3´ and 5´ phosphates from DNA and RNA.

Concentration: 20 units/µL

Applications:
Dephosphorylation of 5´-phosphorylated termini of vector DNA to prevent self-ligation (1). Dephosphorylation of 5´ termini of nucleic acids prior to forward reaction with kinase.

Source: Purified from calf intestinal mucosa.

Performance and Quality Testing:
Endodeoxyribonuclease, 3´ exodeoxyribonuclease, and ribonuclease assays; dephosphorylation efficiency measured in a transformation assay.

Unit Definition:
One unit hydrolyzes 1 µmol of 4-nitrophenyl phosphate in 1 min. at 37°C.

Unit Reaction Conditions:
1 M diethanolamine buffer, 10 mM 4-nitrophenyl phosphate, 0.25 mM MgCl2 (pH 9.8) in 900 µl for 10 min. at 37°C.

DNase I, RNase-free (1 U/µL) (Thermo Scientific™)

Thermo Scientific DNase I, RNase-free is an endonuclease that digests single- and double-stranded DNA. It hydrolyzes phosphodiester bonds producing mono- and oligodeoxyribonucleotides with 5'-phosphate and 3'-OH groups.

The enzyme activity is strictly dependent on Ca2+ and is activated by Mg2+ or Mn2+ ions.

In the presence of Mg2+, DNase I cleaves each strand of dsDNA independently in a statistically random fashion. In the presence of Mn2+, the enzyme cleaves both DNA strands at approximately the same site, producing DNA fragments with blunt-ends or with overhang termini of only one or two nucleotides.

Highlights

• Recombinant enzyme
• Purified from non-animal host with a lower level of intrinsic RNases

Applications

• Preparation of DNA-free RNA
• Removal of template DNA following in vitro transcription
• Preparation of DNA-free RNA prior to RT-PCR and RT-qPCR
• DNA labeling by nick-translation in conjunction with DNA Polymerase I
• Studies of DNA-protein interactions by DNase I, RNase-free footprinting
• Generation of a library of randomly overlapping DNA inserts. Reaction buffer containing Mn2+ is used

Note

DNase I is sensitive to physical denaturation. Mix gently by inverting the tube. Do not vortex.

CIAP (Calf Intestinal Alkaline Phosphatase), 1 U/µL (Invitrogen™)

Calf Intestinal Alkaline Phosphatase (CIAP) is a phosphomonoesterase that removes 3´ and 5´ phosphates from DNA and RNA.

Concentration: 1 unit/µL

Applications:
Dephosphorylation of 5´-phosphorylated termini of vector DNA to prevent self-ligation (1). Dephosphorylation of 5´ termini of nucleic acids prior to forward reaction with kinase.

Source: Purified from calf intestinal mucosa.

Performance and Quality Testing:
Endodeoxyribonuclease, 3´ exodeoxyribonuclease, and ribonuclease assays; dephosphorylation efficiency measured in a transformation assay.

Unit Definition:
One unit hydrolyzes 1 µmol of 4-nitrophenyl phosphate in 1 min. at 37°C.

Unit Reaction Conditions:
1 M diethanolamine buffer, 10 mM 4-nitrophenyl phosphate, 0.25 mM MgCl2 (pH 9.8) in 900 µl for 10 min. at 37°C.

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

DNase I, RNase-free, HC (50 U/µL) (Thermo Scientific™)

Thermo Scientific DNase I, RNase-free is an endonuclease that digests single- and double-stranded DNA. It hydrolyzes phosphodiester bonds producing mono- and oligodeoxyribonucleotides with 5'-phosphate and 3'-OH groups.

The enzyme activity is strictly dependent on Ca2+ and is activated by Mg2+ or Mn2+ ions.

In the presence of Mg2+, DNase I cleaves each strand of dsDNA independently in a statistically random fashion. In the presence of Mn2+, the enzyme cleaves both DNA strands at approximately the same site, producing DNA fragments with blunt-ends or with overhang termini of only one or two nucleotides.

Highlights

• Recombinant enzyme
• Purified from non-animal host with a lower level of intrinsic RNases

Applications

• Preparation of DNA-free RNA
• Removal of template DNA following in vitro transcription
• Preparation of DNA-free RNA prior to RT-PCR and RT-qPCR
• DNA labeling by nick-translation in conjunction with DNA Polymerase I
• Studies of DNA-protein interactions by DNase I, RNase-free footprinting
• Generation of a library of randomly overlapping DNA inserts. Reaction buffer containing Mn2+ is used

Note

DNase I is sensitive to physical denaturation. Mix gently by inverting the tube. Do not vortex.

T4 DNA Ligase (5 U/µL) (Thermo Scientific™)

Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA. The enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids. It also joins DNA fragments with either cohesive or blunt termini, but has no activity on single-stranded nucleic acids.

T4 DNA Ligase requires ATP as a cofactor.

Highlights

• Active in Themo Scientific restriction enzyme, PCR, and RT buffers (when supplemented with ATP)
• Fast—sticky-end ligation is completed in 10 minutes at room temperature
• Supplied with PEG solution for efficient blunt-end ligation

Applications

• Cloning of restriction enzyme generated DNA fragments
• Cloning of PCR products
• Joining of double-stranded oligonucleotide linkers or adaptors to DNA
• Site-directed mutagenesis
• Amplified fragment length polymorphism (AFLP)
• Ligase-mediated RNA detection (see Reference 3)
• Nick repair in duplex DNA, RNA or DNA/RNA hybrids
• Self-circularization of linear DNA.

Includes

• T4 DNA Ligase
• 10X T4 DNA Ligase Buffer
• 50% PEG Solution

Notes

• Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X DNA Loading Dye & SDS Solution at 70°C for 5 min or 65°C for 10 minutes and chill on ice prior to electrophoresis.
• The volume of the ligation reaction mixture should not exceed 10% of the competent cell volume in the transformation process.
• Prior to electro-transformation, remove T4 DNA Ligase from the ligation mixture using spin columns or chloroform extraction. The extracted DNA can be further precipitated with ethanol.

Ambion™ RNase I, cloned, 100 U/µL (Invitrogen™)

Ambion® RNase I efficiently cleaves after all four bases of single-stranded RNA, in contrast to RNase A, which only cleaves after C and U residues. RNase I degrades all RNA dinucleotide bonds leaving a 5' hydroxyl and 2', 3' cyclic monophosphate. Supplied in one tube of 25,000 U (100 U/ µL). RNase I will degrade any RNA to a mixture of mononucleotides, dinucleotides, and trinucleotides and does not degrade DNA, although it will bind to DNA. It has a marked preference for single-stranded RNA over double-stranded RNA, which allows it to work well in ribonuclease protection assays. This RNase I is rigorously tested for nonspecific contaminating endonuclease, exonuclease, and protease activity.

Unit Definition:
One unit is the amount of enzyme required to produce 1 µg of acid-soluble material from mouse liver RNA in 30 min at 37°C.

RNase Cocktail™ Enzyme Mix (Invitrogen™)

Ambion® RNase Cocktail™ is a mixture of two highly purified ribonucleases, RNase A (500 U⁄ml) and RNase T1 (20,000 U⁄ml) and is free of DNase and nicking activities. Use RNase Cocktail for all situations where it is desirable to degrade RNA, i.e. plasmid minipreps and ribonuclease protection assays. RNase Cocktail is supplied in 50% glycerol for maximum convenience. Digestion of RNA with RNase A alone leaves fragments of RNA which are large enough to be visible on agarose gels and precipitate in ethanol. RNase A cuts after C and U residues, and RNase T1 cuts after G residues. Consequently, the mixture of both enzymes results in a reduction in RNA fragment size over the use of either alone.

Quality Control
RNase Cocktail Enzyme Mix is rigorously tested for contaminating nonspecific endonuclease, exonuclease, and protease activity. Functionality is determined in a ribonuclease protection assay.

Unit Definitions:
RNase A: One unit of RNase A is the amount required to give an increase in absorption at 286 nm of 0.0146 absorbance units per minute in a 1 mL volume. Unit assay conditions: 100 mM Tris-acetate (pH 6.5), 1 mM EDTA and 1 mM cyclic 2', 3'-CMP.

RNase T1: 100 Units of RNase T1 is the amount of enzyme that yields an increase in absorption at 260 nm of 0.01428 units per min at room temperature using 60 µg⁄mL yeast total RNA as a substrate.

We also now offer Ambion® RNA-Grade ribonucleases A, V1, and T1 for use in RNA structure⁄function studies. For more information, see: AM2274 RNase A (1 µg⁄ml), AM2275 RNase V1 (0.1 U⁄µl), AM2283 RNase T1 (1 U⁄µl).

Terminal Deoxynucleotidyl Transferase, recombinant (Invitrogen™)

Terminal Deoxynucleotidyl Transferase, Recombinant (rTdT) is a DNA polymerase that catalyzes the addition of deoxynucleotides to the 3fi hydroxyl terminus of DNA. A TdT Technical Bulletin is available.

Applications:
Homopolymer tailing of vector and insert for cloning. Labeling oligonucleotides with biotin (1,2), 32P- or 35S-label (3), or in apoptosis (TUNEL) (4,5).

Source:
Purified from E. coli clone of calf thymus TdT.

Performance and Quality Testing:
Endonuclease, 3´ and 5´ exodeoxyribonuclease, and levels of incorporation tested.

Unit Definition:
One unit incorporates 1 nmol dATP into acid-precipitable material in 1 h at 37°C, using d(pA)50 as a primer.

Hazard Warning:
Toxic; potassium cacodylate contained in reaction buffer. Also contains cobalt chloride, a highly toxic chemical.
See MSDS.

Unit Reaction Conditions:
0.2 M potassium cacodylate (pH 7.2), 10 mM MgO4 C4 H6 , 1 mM 2-mercaptoethanol, 0.5 mg/ml BSA,
100 flM d(pA)50, 1 mM [3H]dATP, and enzyme in 0.15 ml for 1 h at 37°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

T4 DNA Ligase (1 U/µL) (Invitrogen™)

T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes. Single-stranded nucleic acids are not substrates for this enzyme.

Applications
Cloning (blunt-end or cohesive-end ligation) and adding linkers or adapters to blunt-ended DNA

Source
Purified from E. coli lambda lysogen NM989

Performance and quality testing
Endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency tested

Unit definition
One unit catalyzes the exchange of 1 nmol 32P-labeled pyrophosphate into ATP in 20 min at 37°C. One unit is equal to approximately 300 cohesive-end ligation units.

Unit reaction conditions
66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 10 mM DTT, 66 µM ATP, 3.3 µM 32P-labeled pyrophosphate, and enzyme in 0.1 mL for 20 min at 37°C.

TURBO DNA-free™ Kit (Invitrogen™)

The TURBO DNA-free™ Kit contains reagents for the efficient, complete digestion of DNA along with the removal of the enzyme and divalent cations post-digestion.

Note: if you would like to purchase the enzyme alone, without the inactivation and cation removal reagents, please see TURBO™ DNAase.

Features of the TURBO DNA-free™ Kit include:

• Hyperactive TURBO™ DNase is a catalytically superior enzyme compared to wild type DNase I
• Removes trace quantities of DNA that can interfere with RT-PCR
• Reagent included to completely remove DNase without phenol treatment or heating

The best method for genomic DNA removal prior to RT-PCR
TURBO™ DNase is a recombinant, engineered form of DNase I that is much more efficient than wild type DNase I in digesting away trace amounts of unwanted DNA. TURBO™ DNase binds DNA substrates 6-fold more tightly than traditional DNase I, making this enzyme the tool of choice for clearing residual DNA that can generate a false positive signal in RT-PCR applications. TURBO™ DNase now includes an enhancer that increases the effectiveness by two orders of magnitude.

Efficient DNase and divalent cation removal without organic extraction or precipitation
Conventional DNase treatment of RNA samples prior to RT-PCR typically call for inactivation of the DNase by phenol:CHCl3 extraction or heating followed by a precipitation step to concentrate the RNA. Phenol:CHCl3 extractions can be cumbersome and time-consuming. Heating the sample to inactivate DNase can lead to chemical degradation of the RNA by divalent cations present in the DNase buffer. The TURBO DNA-free™ Kit circumvents these problems using a novel DNase Inactivation Reagent. In addition to removing the TURBO™ DNase from the reaction, the Inactivation Reagent also binds and removes divalent cations from the TURBO™ DNase Reaction Buffer.

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.

Terminal Deoxynucleotidyl Transferase (20 U/µL) (Thermo Scientific™)

Thermo Scientific Terminal Deoxynucleotidyl Transferase (TdT) is a template-independent DNA polymerase that catalyzes the repetitive addition of deoxyribonucleotides to the 3'-OH of oligodeoxyribonucleotides and single-stranded and double-stranded DNA. TdT requires an oligonucleotide of at least three nucleotides to serve as a primer. With RNA as template TdT shows variable performance which strongly depends upon the tertiary structure of acceptor RNA 3'-end and the nature of nucleotide. Generally, it is lower than using DNA as a template.

Highlights

Incorporates modified nucleotides (e.g., fluorescein-, biotin-, aminoallyl-labeled nucleotides)

Applications

• Production of synthetic homo- and heteropolymers
• Homopolymeric tailing of linear duplex DNA with any type of 3'-OH terminus
• Oligodeoxyribonucleotide and DNA labeling
• 5'-RACE (Rapid Amplification of cDNA Ends)
In situ localization of apoptosis

Note

Due to the presence of CoCl2, the TdT Reaction Buffer is incompatible with downstream applications. It is necessary to remove CoCl2 from the reaction mixture by spin column or phenol/chloroform extraction and subsequent ethanol precipitation.

S1 Nuclease (100 U/µL) (Thermo Scientific™)

Thermo Scientific S1 Nuclease degrades single-stranded nucleic acids, releasing 5'-phosphoryl mono- or oligonucleotides. It is five times more active on DNA than on RNA. S1 Nuclease also cleaves dsDNA at the single-stranded region caused by a nick, gap, mismatch or loop. S1 Nuclease exhibits 3'-phosphomonoesterase activity.

The enzyme is a glycoprotein with carbohydrate content of 18%.

Applications

• Removal of single-stranded overhangs of DNA fragments
• S1 transcript mapping
• Cleavage of hairpin loops
• Creation of unidirectional deletions in DNA fragments in conjunction with Exonuclease III

Note

S1 Nuclease can introduce breaks into double-stranded DNA, RNA and DNA/RNA hybrids at high enzyme and low salt concentrations.