Shop All DNA⁄RNA Modifying Enzymes

Fast DNA End Repair Kit (Thermo Scientific™)

Thermo Scientific Fast DNA End Repair Kit is used for blunting and phosphorylation of DNA ends in just 5 minutes for subsequent use in blunt-end ligation.

All components of the kit contain premixed reagents to reduce pipetting steps and provide convenience. The End Repair Enzyme Mix contains an optimized mixture of T4 DNA Polymerase and Klenow Fragment to achieve highly effective blunting of fragmented DNA, and T4 Polynucleotide Kinase (PNK) for efficient phosphorylation of DNA ends. The 10X End Repair Reaction Mix contains an optimized reaction buffer, ATP, and dNTPs.

Samples such as fragmented genomic DNA (restriction enzyme digested, nebulized or sonicated), restriction enzyme digested plasmid DNA, double stranded cDNA, and PCR products containing dA overhangs are all compatible with the kit.

Features

During the DNA end repair reaction, fragmented DNA is converted into blunt-end DNA containing a 5'-phosphate and 3'-hydroxyl groups. The 5'→3' polymerase activity of the End Repair Enzyme Mix fills-in 5' protruded DNA ends while 3'→5' exonuclease activity removes 3'-overhangs. T4 PNK adds 5'-phosphates to ends of unphosphorylated DNA fragments, such as PCR products. (see Table 1 and Figure 1 below).

Highlights

Efficient - blunting and phosphorylation of 0.5 to 5 µg DNA
Fast—reaction is completed in 5 minutes
Convenient—reaction components are premixed to reduce pipetting steps

Applications

• Blunting and phosphorylation of double-stranded DNA (nebulized DNA, sonicated DNA, restriction enzyme digested DNA, cDNA, PCR products)

Includes

• End Repair Enzyme Mix
• 10X End Repair Reaction Mix

Related Products
CloneJET PCR Cloning Kit

Uracil-DNA Glycosylase (1 U/µL) (Thermo Scientific™)

Thermo Scientific Uracil-DNA Glycosylase (UDG, UNG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and sugar, leaving an apyrimidinic site in uracil-containing single or double-stranded DNA (see Figure 1 in Supporting Data). The enzyme shows no activity on RNA.

Highlights

Active in Thermo Scientific buffers for restriction enzymes and thermophilic polymerases

Applications

• Control of carry-over contamination in PCR
• Glycosylase mediated single nucleotide polymorphism detection (GMPD)
• Site-directed mutagenesis
• As a probe for protein-DNA interaction studies
• SNP genotyping
• Cloning of PCR products
• Generation of single strand overhangs of PCR products and cDNA

Note

The abasic sites formed in DNA by Uracil-DNA Glycosylase may be subsequently cleaved by heat, alkali-treatment or endonucleases that cleave specifically at abasic sites. UDG (UNG) is active in the presence or absence of divalent cations.

Use of this enzyme in certain applications may be covered by patents and may require a license.

Topoisomerase I (Invitrogen™)

Topoisomerase I (DNA-relaxing enzyme) catalyzes the removal of superhelical turns from covalently closed DNA by a transient breakage and rejoining of phosphodiester bonds. Topoisomerase I is active in the presence of EDTA.

Applications: Relaxing positively and negatively supercoiled DNA (1). Producing DNA topoisomers (2).

Source: Purified from calf thymus.

Performance and Quality Testing: Endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease, and phosphatase assays; conversion of super-coiled DNA to relaxed DNA.

Unit Definition: One unit catalyzes the conversion of 0.5 µg of superhelical Φ X174 RF DNA to a relaxed state in 30 min. at 37°C.

Unit Reaction Conditions: 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2 , 0.1 mM EDTA, 0.5 mM DTT, 30 µg/ml/BSA, 0.5 µg
Φ X174 RF DNA, and enzyme in 50 µl for 30 min. at 37°C.

PureLink™ DNase Set (Invitrogen™)

The PureLink® DNase Set provides rapid and efficient removal of DNA from RNA that has been purified using PureLink® RNA kits. The PureLink® DNase Set is:

• RNase-free for use in RNA purification
• Optimized for on-column digestion
• Lyophilized for stability

Using the PureLink® DNase Set
The RNase-free DNase set comes lyophilized and is optimized for on-column digestion of DNA using PureLink® protocols. The degree of DNA digestion performed by PureLink® DNase is optional for most PureLink® RNA kits, due to the efficient removal of the majority of DNA by the PureLink® technology; however, some downstream applications that are sensitive to small amounts of DNA may require further removal of DNA.

PureLink™ RNase A (20 mg/mL) (Invitrogen™)

PureLink™ RNase A (ribonuclease A) is a bovine pancreatic ribonuclease that cleaves single-stranded RNA. This reagent is also a component of the PureLink™ plasmid purification system.

Concentration
20 mg RNase A/mL in 50 mM Tris-HCl (pH 8.0), 10 mM EDTA.

Anza™ DNA Blunt End Kit (Invitrogen™)

The Invitrogen™ Anza™ DNA Blunt End Kit is used to convert DNA with cohesive ends to DNA with blunt ends for use in blunt-end ligation reactions. The included Anza™ DNA Blunting Enzyme Mix contains T4 DNA polymerase and Klenow Fragment, while the Anza™ 10X Blunting Buffer contains dNTPs to facilitate the synthesis of blunt ends. Use this kit when the desired vector does not contain a recognition site that would produce blunt ends.

Features:
• Fast 15-minute, room-temperature protocol
• DNA digested with Anza™ restriction enzymes can be used directly in the Anza Blunt End protocol following heat inactivation

The Anza DNA Blunt End Kit is part of the Anza™ Restriction Enzyme Cloning System, unifying the traditional cloning processes. Other Anza DNA Modifying Enzymes include the Anza T4 DNA Ligase Master Mix, Anza T4 PNK Kit, and Anza DNA End Repair Kit.

For more information on the Anza system, visit www.thermofisher.com/anza.

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.

Shrimp Alkaline Phosphatase (SAP) (Applied Biosystems™)

Proven Performance – the Phosphatase benchmark
•100% heat-inactivated in 15 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.

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.

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).

Anza™ DNA End Repair Kit (Invitrogen™)

The Invitrogen™ Anza™ DNA End Repair Kit is used to convert DNA with cohesive ends to blunt-ended 5’-phosphorylated DNA for use in blunt-end ligation reactions. The included pre-mixed Anza™ DNA End Repair Mix contains T4 DNA polymerase, Klenow Fragment, and T4 PNK (polynucleotide kinase) to minimize pipetting, while the Anza™ 10X End Repair Buffer contains ATP and dNTPs to facilitate the activity of the enzyme mix.

Benefits:
Convenient—pre-mixed enzyme mixture for superior convenience and reduction of pipetting
Efficient—simultaneous blunting and phosphorylation
Compatible—use directly in Anza restriction enzyme digestion reaction mixture following heat inactivation
Fast—end repair complete in 15 minutes

The Anza DNA End Repair Kit is part of the Anza™ Restriction Enzyme Cloning System, unifying the traditional cloning processes. Other Anza DNA Modifying Enzymes include the Anza T4 DNA Ligase Master Mix, Anza T4 PNK Kit, and Anza DNA Blunt End Kit.

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.

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.

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).

Endonuclease V, T.maritima (5 U/µL) (Thermo Scientific™)

Thermo Scientific Endonuclease V, T. maritima (Endo V) is a 3'-endonuclease involved in DNA repair, which initiates removal of deaminated bases from damaged DNA, including uracil, hypoxanthine, and xanthine.

Endonuclease V is also active toward abasic sites and urea sites, base pair mismatches, flap and pseudo Y structures, and small insertions/deletions in DNA molecules. The cleavage site generated by Endonuclease V is at the second phosphodiester bond 3' to a lesion.

Highlights

• Optimal activity at temperatures of 65 to 70°C

Applications

• High-throughput methods for mutation research
• Studies in mutagenesis and DNA repair
• Mismatch cleavage
• Genotyping

Note

Use of this enzyme in certain applications may be covered by patents and may require a license.

When the enzyme is in excess, the primary nicked products experience a second nicking event on the complementary strand, leading to a double-stranded break. At low concentrations, however, Endonuclease V first nicks a DNA strand at the lesions located closer to the 5'-end of DNA molecule. Single-stranded DNA is cleaved with much lower efficiency. Mg2+ or Mn2+ ions are required for enzyme activity.