Shop All DNA⁄RNA Modifying Enzymes

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.

Micrococcal Nuclease (300 U/µL) Thermo Scientific™

Thermo Scientific Micrococcal Nuclease (S7 Nuclease) is an endo-exonuclease that digests single-stranded and double-stranded DNA and RNA.
Micrococcal Nuclease (S7 Nuclease) is a relatively nonspecific endo-exonuclease that digests single-stranded and double-stranded nucleic acids, but is more active on single-stranded substrates. Cleavage of DNA or RNA occurs preferentially at AT or AU-rich regions, yielding mononucleotides and oligonucleotides with terminal 3'-phosphates. The enzyme's activity is strictly dependent on Ca2+.
Applications

• Hydrolysis of nucleic acids in crude cell-free extracts
• Sequencing of RNA (see​ Reference 2)
• Studies of chromatin structure
• A model for protein folding and for structure-function studies

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) 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 H, from E. coli, 10 U/µL Invitrogen™

Ambion® RNase H (Ribonuclease 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 1,000 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.

RNase A Thermo Scientific™

Thermo Scientific GeneJET RNase A Solution is a component of the GeneJET Plasmid Miniprep Kit (K0502/K0503) and may be purchased separately.

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.

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).

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.

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.

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.

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

RNase H (5 U/µL) Thermo Scientific™

Thermo Scientific Ribonuclease H (RNase H) specifically degrades the RNA strand in RNA-DNA hybrids. It does not hydrolyze the phosphodiester bonds within single-stranded and double-stranded DNA and RNA.

Applications

• Removal of mRNA prior to synthesis of second strand cDNA
RT-PCR and qRT-PCR: removal of RNA after first strand cDNA synthesis
• Removal of the poly(A) sequences of mRNA after hybridization with oligo(dT)
• Site-specific cleavage of RNA
• Studies of in vitro polyadenylation reaction products

HotStart-IT Binding Protein Thermo Scientific™

Application
Hot-Start PCR Amplification

HotStart-IT™ Binding Protein is the active component in a novel hot start technology called primer sequestration. In general, hot start PCR methods reduce or eliminate non-specific primer-extension products formed at lower temperatures during assembly of PCR reactions. At these less stringent annealing temperatures, primers may bind non-specifically, which often leads to unwanted amplification products and primer-dimers. In order to resolve this problem, we have produced a high-quality DNA binding protein that is especially useful at sequestering primers at lower temperatures making them unavailable for use by a polymerase (Fig. 1). This primer-sequestration technique effectively blocks DNA synthesis from mis-priming events at lower temperatures. Following the initial denaturation step during PCR, the protein is inactivated and the primers are free to participate in the amplification reaction. Unlike other hot start methods, such as antibodies or chemical modifications, the binding protein is compatible with a variety of thermostable polymerases. HotStart-IT Binding Protein has been designed for PCR applications that demand extremely high specificity and sensitivity and is thoroughly tested for purity and performance.

Properties
HotStart-IT Binding Protein performs well in many standard PCR reaction buffers.

Purity
Free from detectable non-specific nucleases.

Storage Buffer
20 mM Tris-HCl (pH 8.5), 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol.

Mass Definition
In general, one microgram of HotStart-IT Binding Protein sequesters about 5 pmol of primers.

Concentration
10 ± 0.5 mg/mL

Source
Recombinant protein expressed in E. coli.

Advantages of HotStart-IT
• Room temperature reaction set-up
• High specificity and sensitivity
• Minimizes amplification of non-specific products and primer-dimers (Fig. 2)
• Ideal for complex templates and multiplex reactions
• Unlike chemically-modified Taq, no extensive heating step is necessary which may damage precious samples
Technology is portable to a polymerase of choice.

Quality Control Polymerase Blocking Assay
This assay compares the amount of Taq DNA Polymerase activity with 2 µg HotStart-IT Binding Protein against its activity with no binding protein. The reaction mixtures contain 0.625 units of polymerase, 1X PCR Reaction Buffer (10 mM Tris-HCl [pH 8.6], 50 mM KCl, 1.5 mM MgCl2), 0.2 mM each dNTP, and 2 pmol of overlapping, extendable oligonucleotides in a 25 µL reaction volume. Following incubation at 25 °C for 4 hours, HotStart-IT Binding Protein blocks at least 90% of the Taq DNA Polymerase activity. (See Fig. 2 for representative data.)

Functional Test
PCR with Taq DNA Polymerase and HotStart-IT Binding Protein shifts production of primer-dimers to a specific target of 306 bp from 1 ng of human genomic DNA relative to Taq DNA Polymerase alone.

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
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