Shop All DNA⁄RNA Modifying Enzymes

Ribonuclease H (Invitrogen™)

Ribonuclease H (RNase H) is an endoribonuclease that specifically degrades the RNA strand of an RNA-DNA hybrid to produce 5´ phosphate-terminated oligoribonucleotides and single-stranded DNA.

Applications

Removal of mRNA during second-strand cDNA synthesis. Removal of poly(A) sequences from mRNA in the presence of oligo(dT). Oligodeoxyribonucleotide-directed cleavage of RNA.

Source

Purified from E. coli expressing the E. coli RNase H gene on a plasmid.

Performance and quality testing

Ribonuclease, nonspecific endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease.

Unit definition

One unit hydrolyzes 1 nmol of RNA in 3H-labeled poly(A)poly(dT) to acid-soluble material in 20 min at 37°C.

Unit reaction conditions

20 mM Tris-HCl (pH 7.5), 0.1 M KCl, 10 mM MgCl2 , 0.1 mM DTT, 5% (w/v) sucrose, 0.5 nmol 3H-labeled poly(A)poly(dT), and enzyme in 50 µL for 20 min at 37°C.

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), 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.

Ambion™ Recombinant RNase A (Invitrogen™)

Ambion® Recombinant RNase A for the production of plasmid DNA that is substantially free of host RNA is now available for research applications. For large batch sizes, the use of RNase is still the only effective technique applicable to remove host RNA. Other methods such as selective precipitation, gel filtration, chromatography, and endogenous E. coli RNase digestion have only been effective for small-scale or bench-scale purification. This new product was designed specifically to minimize the risk of introducing pathogens into bioprocessing. Recombinant RNase A is rigorously tested for contaminating nonspecific endonuclease, exonuclease, and protease activity.

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

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.

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.

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.

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.

FastAP Thermosensitive Alkaline Phosphatase (1 U/µL) (Thermo Scientific™)

Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase catalyzes the release of 5'- and 3'-phosphate groups from DNA, RNA, and nucleotides. This enzyme also removes phosphate groups from proteins.

FastAP is a novel alkaline phosphatase, which is active in all Thermo Scientific restriction enzyme buffers as well as in PCR buffers. It dephosphorylates all types of DNA ends (blunt, 5'- and 3'-overhangs) in 10 minutes at 37°C. The enzyme is inactivated in 5 minutes at 75°C (see Figure 1 in Supporting Data). Therefore, removal of alkaline phosphatase is not required prior to ligation.

Highlights

Recombinant enzyme
Fast dephosphorylation—10 minutes at 37°C
Fast and complete inactivation—5 minutes at 75°C
Simultaneous digestion and dephosphorylation of vector DNA
100% active in restriction enzyme and PCR buffers
PCR clean-up in conjunction with Exo I
Protein dephosphorylation

One protocol for all types of DNA ends:
• 5'-overhangs
• 3'-overhangs
• blunt-ends
• single nucleotides

Applications

• Dephosphorylation of cloning vector DNA to prevent recircularization during ligation
• Simultaneous digestion and dephosphorylation of vector DNA
• PCR product clean-up: nucleotide degradation prior to sequencing of PCR product
• Dephosphorylation of nucleic acid 5'-termini prior to labeling with T4 Polynucleotide Kinase
• Other applications where dephosphorylation of DNA and RNA substrates is necessary
• Protein dephosphorylation

Note

• Binding of FastAP Thermosensitive Alkaline Phosphatase to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X DNA Loading Dye & SDS Solution at 65°C for 10 minutes, and chill on ice prior to electrophoresis.
• FastAP Thermosensitive Alkaline Phosphatase is active in all restriction enzyme buffers and may be added directly to digested DNA. Heat inactivation of the restriction enzyme before dephosphorylation reaction is not necessary.

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.

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.

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).