Shop All DNA⁄RNA Modifying Enzymes

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.

T4 DNA Ligase, LC (1 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.

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.

T4 RNA Ligase (10 U/µL) (Thermo Scientific™)

Thermo Scientific T4 RNA Ligase catalyzes the ATP-dependent intra- and intermolecular formation of phosphodiester bonds between 5'-phosphate and 3'-hydroxyl termini of oligonucleotides, single-stranded RNA and DNA.

The minimal substrate is a nucleoside 3',5'-biphosphate in intermolecular reaction and oligonucleotide of 8bases in intramolecular reaction.

Applications

• RNA 3'-end labeling with cytidine 3',5'-bis [alpha-32P] phosphate
• Joining RNA to RNA
• Synthesis of oligoribonucleotides and oligodeoxyribonucleotides
• Specific modifications of tRNAs
• Oligodeoxyribonucleotide ligation to single-stranded cDNAs for 5' RACE (Rapid Amplification of cDNA Ends)
• Site-specific generation of composite primers for PCR

Note

The recommended BSA concentration in the reaction mixture is 0.1mg/mL.

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 1 mg (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 DNA Ligase (5 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 (1). Single-stranded nucleic acids are not substrates for this enzyme. A T4 DNA Ligase Technical Bulletin is available.

Applications: Cloning (blunt-end or cohesive-end ligation) (2). Adding linkers or adapters to blunt-ended DNA (2).

Source: Purified from E. coli œ 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 32 P-labeled pyrophosphate, and enzyme in 0.1 ml for 20 min. at 37°C.

AmpErase™ Uracil N-Glycosylase (UNG) (Applied Biosystems™)

AmpErase® Uracil N-Glycosylase (UNG), part of the GeneAmp® PCR Carry-over Prevention Kit, is a 26 kDa ultrapure, recombinant enzyme encoded by the E. coli uracil N-glycosylase gene, which has been inserted into an E. coli host to direct the expression of the wild type form of the enzyme. The enzyme removes any uracil incorporated into single- or double-stranded DNA.

GeneAmp® PCR Carry-over Prevention Kit
The GeneAmp® PCR Carry-over Prevention Kit (available separately) provides reagents for a simple yet powerful method of ensuring that PCR products cannot be reamplified in subsequent PCR amplifications, thereby preventing false positive results. Features of this kit:

• Enzymatic method stops PCR carryover contamination, which prevents false positive results
• Designed to degrade PCR products from previous PCR amplifications without degrading native nucleic acid templates, which improves amplification
• No interference with any PCR or real-time PCR application
• Optimized for use with GeneAmp® PCR core reagents and GeneAmp® instrument systems

Eliminate PCR carryover contamination
The kit uses an enzymatic reaction analogous to the restriction-modification and excision-repair systems of cells to specifically degrade PCR products from previous PCR amplifications in which dUTP has been incorporated, without degrading native nucleic acid templates. The method used to render PCR products susceptible to degradation involves substituting dUTP for dTTP in the PCR mixture, and pretreating all subsequent PCR mixtures with AmpErase® Uracil N-glycosylase (UNG) prior to PCR amplification. Products from PCR amplification contain uracil, and are readily distinguishable from native thymidine-containing DNA templates. Products from previous PCR amplifications are eliminated by excising uracil residues using UNG, and degrading the resulting abasic polynucleotide with heat. Although UNG is active on single- and double-stranded dU-containing DNA, ribouracil residues in RNA and dUTP itself are not substrates for UNG. AmpliTaq® DNA Polymerase, AmpliTaq Gold® DNA Polymerase, and the other components of the PCR mixture remain intact during UNG treatment, providing PCR amplification free of PCR product carryover. The dU-containing PCR product behaves like dT-containing DNA in blotting, cloning, sequencing, and most other post-PCR analyses.

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.

Exonuclease III (200 U/µL) (Thermo Scientific™)

Thermo Scientific Exonuclease III (ExoIII) exhibits four catalytic activities. The 3'→5' exodeoxyribonuclease activity of ExoIII is specific for double-stranded DNA. ExoIII degrades dsDNA from blunt ends, 5'-overhangs or nicks, releases 5'-mononucleotides from the 3'-ends of DNA strands and produces stretches of single-stranded DNA. It is not active on 3'-overhang ends of DNA that are at least four-bases long and do not carry a 3'-terminal C-residue on single-stranded DNA, or on phosphorothioate-linked nucleotides.

ExoIII 3'-phosphatase activity removes the 3'-terminal phosphate, generating a 3'-OH group. ExoIII Rnase H activity exonucleolytically degrades the RNA strand in RNA-DNA hybrids. ExoIII apurinic/apyrimidinic-endonuclease activity cleaves phosphodiester bonds at apurinic or apyrimidinic sites to produce 5'-termini that are base-free deoxyribose 5'-phosphate residues.

Highlights

• Active in restriction enzyme buffers

Applications

• Creation of unidirectional deletions in DNA fragments in conjunction with S1 Nuclease
• Generation of a single-stranded template for dideoxy-sequencing of DNA
• Site-directed mutagenesis
• Cloning of PCR products
• Preparation of strand-specific probes

Note

The rate of DNA digestion by ExoIII depends upon temperature, salt concentration, and the molar ratio of DNA to enzyme in the reaction mixture . Optimal reaction conditions should be determined experimentally.

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 T1 (1 U/μL) (Invitrogen™)

Optimized for researchers performing RNA structure, RNA sequencing, protein footprinting, and boundary experiments, Ambion® RNase T1, RNA-Grade cleaves 3' of single-stranded G residues and is tested for purity to ensure that no contaminating ribonuclease activities are present that could cleave at unanticipated sites. Supplied in one tube of 200 µL (1U/ µL). RNase T1 can be used to perform boundary experiments to define the minimal RNA sequence required for selectable activities such as protein binding or catalysis. In addition to applications for RNA structural analysis, RNA-Grade ribonucleases can be used to map protein binding sites on RNAs by comparing cleavage patterns in the presence and absence of an RNA binding protein.

Unit Definitions:
100 units 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.

Accessory Products:
Other RNA-Grade ribonuclease available include RNase A (SKU# AM2274), which cleaves 3' of single-stranded C and U residues. Combinations of the single- and double-stranded specific ribonucleases can provide rapid analysis of the physical structure of an interesting RNA.

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.

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.

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.

Exonuclease I, high concentration, (20 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), 10mM
2-mercaptoethanol, 6.7mM MgCl2, 0.5mM 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.