Shop All Modifying Enzymes

T4 Polynucleotide Kinase (10 U/µL) Thermo Scientific™

Thermo Scientific™ T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the gamma-phosphate from ATP to the 5'-OH group of single- and double-stranded DNAs and RNAs, oligonucleotides, or nucleoside 3'-monophosphates (forward reaction). The reaction is reversible. In the presence of ADP, T4 Polynucleotide Kinase exhibits 5'-phosphatase activity and catalyzes the exchange of phosphate groups between 5'-P-oligo-polynucleotides and ATP (exchange reaction). The enzyme is also a 3'-phosphatase.

Highlights
• Active in Thermo Scientific restriction enzyme, RT, and T4 DNA ligase buffers

Applications
• Labeling 5' -termini of nucleic acids to be used as:
    --Probes for hybridization
    --Probes for transcript mapping
    --Markers for gel electrophoresis
    --Primers for DNA sequencing
    --Primers for PCR
• 5'-phosphorylation of oligonucleotides, PCR products, other DNA or RNA prior to ligation
• Phosphorylation of PCR primers
• Detection of DNA modification by the [32P]-postlabeling assay
• Removal of 3'-phosphate groups

Notes
• The 5'-termini of nucleic acids can be labeled by either the forward or the exchange reaction.
• Polyethylene glycol (PEG) and spermidine improve the rate and efficiency of the phosphorylation reaction. PEG is used in the exchange reaction mixture.
• Since T4 Polynucleotide Kinase is inhibited by ammonium ions, use sodium acetate to precipitate DNA prior to phosphorylation.

LCK Recombinant Human Protein

LCK is a SRC-related kinase family expressed in lymphocytes.

Anza™ T4 DNA Ligase Master Mix Invitrogen™

The Invitrogen Anza™ T4 DNA Ligase Master Mix facilitates the joining of abutted 5’-phosphate and 3’-hydroxyl termini in duplex DNA through the formation of a phosphodiester bond. It can be used to join DNA fragments with both sticky ends and blunt ends, and to repair nicks in double-stranded DNA with 3'-hydroxyl and 5'-phosphate ends. Anza T4 DNA Ligase is formulated as a 4X concentrated master mix. Ligation can be performed with DNA in water, TE, elution buffer, or 1X Anza™ buffers.

Benefits:
• Ligation complete in 15 minutes at room temperature
• A single ligase master mix for both sticky-end and blunt-end ligation reactions
• Ready-to-use master mix format reduces pipetting steps
• 4X concentration enables more dilute DNA to be used in ligation reaction without concentrating

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

RapidOut DNA Removal Kit Thermo Scientific™

Thermo Scientific RapidOut DNA Removal Kit rapidly and safely removes genomic DNA from total RNA and mRNA preparations. Complete digestion of DNA and safe removal of DNase I from the digestion reaction is ensured without RNA damaging steps, such as heating or organic extraction. First, the RNA sample is treated with recombinant RNase-free DNase I to levels below the limit of detection by routine PCR. DNase I is safely removed subsequently using proprietary DNase Removal Reagent (DRR).

DRR efficiently binds DNase I and the complex is collected at the bottom of the tube by centrifugation. The purified RNA is collected as a supernatant. The RNA after RapidOut procedure is free from DNA contamination and free of DNase I. It is ready to use in different applications including end-point or real-time RT-PCR, cloning, microarrays, and Northern blotting.

Highlights

Efficient—complete ds and ssDNA digestion and proprietary technology for DNase I removal
Rapid—single step sufficient for complete DNase I removal
Safe—no need for toxic organic extractions or RNA-damaging heating steps

Applications

Main Applications
• RNA isolation and RNA analysis, particularly RT-qPCR and RT-PCR (customers performing expression analysis of low transcription level genes.
• Customers performing ds-cDNA synthesis from total RNA preps.

Other applications
• Elimination of DNA from RNA for microinjections and transfection.
• Elimination of DNA from RNA prior microarray analysis.
• Elimination of DNA from RNA prior Northern blot analysis.

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).

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.

Bovine Serum Albumin Invitrogen™

Bovine Serum Albumin is suitable for use as an enzyme stabilizer during purification or for dilution of restriction endonucleases and nucleic acid modifying enzymes. BSA is also commonly used in DNA and protein labeling experiments as a blocking agent to minimize background. This BSA is acetylated to inactivate contaminating nucleases and proteases.

Performance and quality testing
Purity is determined by SDS-PAGE. No detectable contaminating activity is observed in 3´ and 5´ exonuclease, DNA nicking, and restriction endonuclease inhibition assays.

Anza™ Alkaline Phosphatase Invitrogen™

Invitrogen™ Anza™ Alkaline Phosphatase is intended for use in the removal of 3' and 5' phosphate groups that remain after Anza™ restriction enzyme digestion, such as for the dephosphorylation of vectors prior to insert ligation and removal of 5′ phosphates prior to end-labeling. Since the 5′ phosphate group is required for DNA ligation, treatment of vectors with Anza Alkaline Phosphatase prevents self-ligation and re-circularization, resulting in decreased vector background when cloning.

Features of Anza Alkaline Phosphatase:

• Validated for use with all Anza restriction enzymes
• Dephosphorylates in 15 minutes at 37°C
• Heat inactivated after 5 minutes at 80°C
• Fully active in Anza Buffer
• Dephosphorylates all types of DNA ends (blunt, 5'- and 3'-overhangs)

Flexible protocol
Some restriction enzymes prohibit or reduce the dephosphorylation process when digestion and dephosphorylation are performed simultaneously. Therefore, Anza Alkaline Phosphatase may be used in one of three ways depending upon the restriction enzyme used:

One-Step Protocol: Simultaneous restriction enzyme digestion and dephosphorylation in Anza buffer
Heat Inactivation Two-Step Protocol: Digestion is carried out first, followed by heat inactivation (20 minutes at 80oC) then dephosphorylation
Column Purification Two-Step Protocol: Digestion is carried out first, followed by column purification then dephosphorylation

No need for enzyme removal
Anza Alkaline Phosphatase is completely inactivated after 5 minutes at 80°C, so enzyme removal via phenol/chloroform extraction or column purification is not required prior to subsequent ligation steps. If the One-Step protocol was used, heat the reaction mixture for 20 minutes at 80oC to inactivate the restriction enzyme as well as the alkaline phosphatase. Heat inactivation of the alkaline phosphatase prevents residual phosphatase from dephosphorylating the DNA fragments added to the reaction mixture for ligation.

Anza™ Alkaline Phosphatase 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, Anza DNA Blunt End Kit, and Anza DNA End Repair Kit.

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

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.

TURBO™ DNase (2 U/µL) Invitrogen™

TURBO™ DNase cleaves double-stranded DNA nonspecifically to leave 5' phosphorylated oligodeoxynucleotides. It has increased affinity for DNA-binding and remains active in the presence of salt.

Note: this product is just the enzyme. If you would like this enzyme plus reagents to inactivate the enzyme and remove divalent cations post-digestion, please see TURBO DNA-free™ Kit.

Features of TURBO™ DNase include:

• Up to 50x more activity and 350% greater catalytic efficiency
• Efficiently degrades DNA in solutions containing up to 0.25 M salt
• Efficiently digests DNA to oligonucleotides
• Vastly superior in clearing DNA templates from in vitro transcription reactions
• RNase-free and recombinant in origin

Using TURBO™ DNase
DNase I is commonly used to clear DNA contamination from RNA samples prior to RT-PCR. Conventional DNase I has a poor affinity for DNA and cleaves DNA of low concentration very inefficiently. In addition, DNase I is very salt-sensitive; as little as 20 mM NaCl can reduce the activity of the enzyme by 30%. Finally, DNase I is purified from bovine pancreas, one of the richest natural sources of RNase A. The threat of contaminating RNase activity in DNase I preparations requires that the enzyme be exhaustively purified. In spite of these limitations, the DNase I that researchers use today is the very same enzyme that was first characterized by Kunitz more than a half-century ago.

A different DNase with superior properties to wild-type DNase I
TURBO™ DNase was developed using a protein engineering approach that introduced amino acid changes into the DNA binding pocket of wild-type DNase I. These changes markedly increase the affinity of the protein for DNA. The result is a versatile enzyme that has a 6-fold lower Km for DNA, and an ability to maintain at least 50% of peak activity in solutions approaching 200 mM monovalent salt, even when the DNA concentration is in the nanomolar (nM) range. When in vitro transcription reactions are treated with either DNase I or TURBO™ DNase, TURBO™ DNase removes 63x more of the input plasmid DNA template than the wild-type enzyme. The proficiency of TURBO™ DNase in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination. This becomes important for complete removal of DNA from a sample, since the cleavable DNA substrate is reduced as the DNase reaction proceeds. TURBO™ DNase thus has a functional advantage over wild-type DNase due to its superior affinity for DNA. This is best exploited in RT-PCR applications, where even a few copies of DNA can lead to a false positive outcome by PCR.

MAP3K8 (COT) Recombinant Human Protein Thermo Scientific™

MAP3K8 (COT) is a serine⁄threonine kinase which can activate both the MAP and JNK kinase pathways and plays a role in the cell cycle.

DNase I, Amplification Grade Invitrogen™

DNase I, Amplification Grade, digests single- and double-stranded DNA to oligodexyribonuleotides containing a 5' phosphate. DNase I, Amplification Grade, is suitable for eliminating DNA during critical RNA purification procedures such as those prior to RNA-PCR amplification. It is purified and tested for non-detectable levels of RNase contamination. Absence of RNase is tested by performing a ribonuclease assay with RNA ladder.

Application
Removing DNA from RNA and protein preparations.

Specific activity
Specific activity is >10,000 units/mg.

Source
Purified from bovine pancreas

Performance and quality testing
Ribonuclease assay with RNA ladder and ability to digest single-stranded and double-stranded DNA to oligonucleotides are determined.

Unit definition
One unit increases the absorbance of a high molecular weight DNA solution at a rate of 0.001 A260 units/min/mL of reaction mixture at 25°C.

Unit reaction conditions
0.1 M sodium acetate (pH 5.0), 5 mM MgCl2, 50 µg/mL calf thymus DNA, and enzyme in 1 mL for 10 min at 25°C.

TrueCut™ Cas9 Protein v2 Invitrogen™

Invitrogen TrueCut Cas9 Protein v2 is a next-generation CRISPR Cas9 protein engineered to deliver maximum editing efficiency. Features include:

• Consistently high editing efficiency in all tested cell lines including standard, immune, primary, and stem, with up to 2X higher editing efficiency in difficult targets than the competition
• High quality—manufactured under strict ISO 13485 quality standards
• Validated protocols for a large number of cell types help you achieve success faster

TrueCut Cas9 Protein v2 is recombinant Streptococcus pyogenes Cas9 (wt) protein, purified from E. coli, for genome editing with CRISPR technology. Cas9 protein forms a very stable ribonucleoprotein (RNP) complex with the guide RNA (gRNA) component of the CRISPR/Cas9 system. Incorporation of nuclear localization signals (NLS) aids delivery to the nucleus, increasing the rate of genomic DNA cleavage.

• Transfection-ready, using either lipid-mediated transfection reagents or electroporation
• Eliminates time-consuming cloning steps
• Available in 2 concentrations:
    --1 μg/μL for use in standard editing scenarios
    --5 μg/μL for optimization of editing conditions in more difficult scenarios such as in primary or embryonic cells, microinjection, or when screening multiple gRNA sequences at a time
    --For custom sizes or concentrations, please inquire

Options for obtaining CRISPR gRNA for use with TrueCut Cas9 Protein v2:
1. Order transfection-ready TrueGuide synthetic gRNAs
2. Generate transfection-ready gRNA in as little as four hours including template assembly with our GeneArt Precision gRNA Synthesis Kit
3. Have our Custom Services team design, synthesize, and purify in vitro (IVT) gRNA sequences for you

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.

Ambion™ RNase III Invitrogen™

Ambion® Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded RNA (dsRNA) specific endoribonuclease. In E. coli, RNase III cleaves ribosomal RNA (rRNA) precursors during maturation of rRNA. The enzyme cleaves dsRNA into 12–15 bp dsRNA fragments with 2 to 3 nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl termini. The termini and overhangs of RNase III cleavage products are thus the same as those produced by Dicer in the eukaryotic RNAi pathway. Transfection of RNase III cleavage products can be used to induce RNAi in mammalian cells (patent pending).

Unit Definition:
One unit is defined as the amount of enzyme catalyzing the cleavage of 1 µg of 500 bp dsRNA substrate to approximately 12–30 bp fragments in 60 min at 37°C.
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