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Ambion RNase I, cloned, 100 U/L
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 10,000 U (100 U/ L). 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.
One unit is the amount of enzyme required to produce 1 g of acid-soluble material from mouse liver RNA in 30 min at 37C.
Terminal Deoxynucleotidyl Transferase (rTdT), Recombinant
Terminal Deoxynucleotidyl Transferase (TdT) catalyzes the template-independent addition of unlabelled or radio-, fluorescent-, biotin-, or digoxygenin-labelled deoxynucleotides, dideoxynucleotides, or ribonucleotides to the 3-hydroxyl termini of single or double-stranded DNA. TdT requires an oligodeoxynucleotide primer with at least three phosphate groups and a free 3-hydroxyl end, and a divalent cation for activity. TdT also catalyzes the depolymerization of DNA (pyrophosphorolysis) and pyrophosphate exchange. Historically, TdT is used to label DNA probes (either non-isotopic or radiolabeled), in apoptosis detection and quantification in the terminal deoxynucleotidyl transferase dUTP nick end labeling method (TUNEL) (also referred to as ISEL,in situend labeling), in DNA sequencing, and to create sticky ends by 3 tailing DNA prior to annealing and transformation. For 5 RACE, TdT is used to add homopolymer tails to the 3-end of the first strand cDNA. Recently, TdT has been used to clone unknown sequences adjacent to known sequences in genomic DNA and for TdT-dependent PCR(TDPCR). TDPCR is used in RNA analysis, transcription analysis, DNA damage analysis, and DNA footprinting.
Tested for contaminating exonucleases and endonucleases.
50mM potassium phosphate, pH 6.4, 100mM NaCl, 1mM DTT, 0.1% Tween® 20 and 50% glycerol.
The reaction mixture (50 L) containing 25mM sodium cacodylate (pH 7.2), 0.5mM radiolabelled dATP (100 – 150 cpm/pmol), 10mM MgCl2, 0.1mM -mercaptoethanol, 0.01mM
d(pA)35, and enzyme is incubated at 37C for varying amounts of time. DNA is captured and incorporated counts determined.
One unit is the amount of enzyme required to polymerize 1 nmol of dATP residues into DNA in 60 minutes at 37C under standard assay conditions.
Functionally tested by 3-end labeling of a fluorescently labeled oligonucleotide.
Functionally Tested 5X TdT Reaction Buffer (1 ml included, PN 72035):
500mM sodium cacodylate, pH 6.8, 5mM cobalt chloride and 0.5mM DTT.
2. Creating isotopic, fluorescent, biotinylated, or digoxygenin labeled probes.
3. Creating fluorescent 3 labeled sequencing primers.
4. Elimination of background bands in manual DNA sequencing.
6. Detection of apoptotic DNA damage (TUNEL assay).
Invitrogen ezDNase Enzyme is a recombinant double-strand-specific DNase for the fast removal of contaminating genomic DNA from RNA preparations. It cleaves phosphodiester bonds in double-stranded DNA to yield 28 bp oligonucleotides with 5-phosphate and 3-hydroxyl termini.
Efficient and fast genomic DNA removal
Highly specificno impact on RNA, cDNA, or primers in RT reactions
ezDNase Enzymes high specificity for double-stranded DNA enables efficient and fast genomic DNA removal without reduction in the quality or quantity of RNA or single-stranded DNA present in the reaction such as cDNA and primers. ezDNase Enzyme is heat-labile and so can be easily deactivated by heat treatment at moderate temperature (55C). These features make ezDNase a superior choice for genomic DNA removal prior to reverse transcription reactions.
When used in combination with theSuperScript IV VILO Master Mixor other Invitrogen reverse transcription reagents, ezDNase Enzyme helps significantly reduce the possibility of cDNA synthesis being compromised by genomic DNA and reduce variation in RT-qPCR quantitation caused by RNA loss or damage during conventional DNase treatment.
Uracil-DNA Glycosylase, heat-labileUracil-DNA Glycosylase excises uracil from dU-containing DNA by cleaving the N-glycosidic bond between the uracil base and the sugar backbone. This cleavage generates alkali sensitive apyrimidinic sites that are blocked from replication by DNA polymerase or prevented from becoming a hybridization site.
Double and single-stranded dU-containing DNA are substrates for Uracil-DNA Glycosylase whereas RNA and normal dT-containing DNA are not.
Uracil-DNA Glycosylase can also be used to increase the cloning efficiency of PCR products having dU-containing primers incorporated into them as well as increasing the efficiency of site-directed mutagenesis.
The Uracil-DNA Glycosylase derived fromGadus morhuahas all the attributes of the enzyme derived fromE. coliwith the added benefit of being heat-labile. It is completely and irreversibly inactivated after 10 minute incubation at 50C.
The enzyme is chromatographically purified and tested for contaminating endonucleases and exonucleases.
50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.1% Triton X-100,50% glycerol.
The reaction mixture contains 70 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 100 g/mL BSA,3H-dUTP labeled DNA, and Uracil-DNA Glycosylase. Incubation is at 37C for 1 hour.
One unit is the amount of enzyme required to liberate 1 nmol uracil from dU-containing DNA in one hour at 37C.
1.Study of DNA repair and mutation detection.
3.Increase the efficiency of site-directed mutagenesis.
4.Study of protein-DNA interactions.
Recombinant fromGadus morhua(cod liver)
Thermo Scientific transposon products are based on the transposition machinery of the bacteriophage Mu. During the lytic phase of the phages life cycle the machinery replicates its genome by transposing repeatedly inside the host genome. The Mu transposition reaction has been modified into an in vitro reaction catalyzed by a single enzyme – MuA Transposase. In this system, one in vitro reaction is capable of generating more than a million transposon insertion clones.
The Mutation Generation System (MGS Kit) and Stop Generation System (STOP Kit) were developed for functional analysis of proteins. These new transposon tools enable the creation of saturated libraries of mutated proteins in a single reaction with less hands-on time than any other method. The location of the transposon insertion in each mutated clone can be mapped by either PCR or sequencing. With MGS and STOP kits, thousands of mutated clones are ready for expression studies in just 2 to 3 days.
The MGS Kit contains the complete set of reagents for transposon-based linker scanning mutagenesis of any target protein. The MGS Entranceposons are designed for making subtle changes in the structure of a target protein by inserting 15 bp in-frame linkers throughout the corresponding target gene. This in-frame insertion allows for conservation of downstream sequences.
The STOPKit Entranceposons contain translational stop codons in all three reading frames within the terminal portion of the transposon sequence. The proprietary modification of the Stop Generation System makes it possible to generate a saturated C-terminal deletion library from virtually any target protein with a maximum addition of three amino acids.
EfficientCreate saturated insertion libraries for sequencing and protein analysis in a single reaction
FastDecrease hands-on time compared to conventional methods
RandomEliminate target site preference or insertion hot-spot
The STOP Kit generates truncated proteins for functional assays of:
The MGS Kit generates random fifteen basepair in vitro insertions into any target DNA for:
Rapid generation of in-frame five amino acid insertion libraries of any protein for functional analyses
Rapid and random mutagenesis of cloned promoters and other regulatory DNA regions
Random insertion of a NotI restriction enzyme site into any target DNA clone
Thousands of different insertion clones from a single reaction
Generates random insertions of 5 amino acids in all 3 reading frames
Short in-frame insertions; no stop codons
Flexibility in mapping mutants of interest: mutations are easily mapped by NotI or PCR
Faster and more effective than linker scanning mutagenesis
Saturated library of truncated proteins from a single reaction in two days
Translational STOP codon in all three reading frames
The target DNA sequence can be unknown
Faster and more effective than conventional methods
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).
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 g of superhelical X174 RF DNA to a relaxed state in 30 min. at 37C.
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 g/ml/BSA, 0.5 g
X174 RF DNA, and enzyme in 50 l for 30 min. at 37C.
Proven Performance the Phosphatase benchmark
100% heat-inactivated in 5 min at 65C
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 fromPandalus 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 65C 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.
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.
Heat-Inactivation: 65C for 15 min.
Inhibitors: 10mM DTT, 0.1% -ME
Reaction Conditions: Active in NaCl, KCl. Requires Mg2+for highest activity.
Tested for contaminating endonucleases, exonucleases, and ribonucleases.
25mM Tris-HCl (pH 7.5), 1mM MgCl2, 50% glycerol.
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 L reaction volume).
One unit is the amount of enzyme which catalyzes the hydrolysis of 1 mol of p-nitrophenyl phosphate per min in glycine buffer (pH 10.4) at 37C.
Dephosphorylation ofrestriction 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 theUSB 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):
1. RUAN, C. C., SAMOLS, S. B. AND FULLER, C. W. (1990)Comments17,(No.1), United States Biochemical Corporation, Cleveland, OH.
2. WERLE, E., SCNEIDER C., RENNER, M., VLKER, M. AND FIEHN, W. (1994)Nucleic Acids Res.22, 4354-4355.
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.
Efficientcomplete ds and ssDNA digestion and proprietary technology for DNase I removal
Rapidsingle step sufficient for complete DNase I removal
Safeno need for toxic organic extractions or RNA-damaging heating steps
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.
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.
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
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.
Bacterial Alkaline Phosphatase (BAP) removes 3 and 5 phosphates from DNA and RNA. BAP is active at 65C for at least 1 h and is inactivated by phenol extraction.
Applications:Dephosphorylation of 5-phosphorylated termini of vector DNA to prevent self-ligation. Dephosphorylation of 5 termini of nucleic acids prior to forward reaction with kinase.
Performance and Quality Testing:Endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease assays; dephosphorylation
Unit Definition:One unit hydrolyzes 1 nmol of ATP in 30 min. at 37C.
10 mM Tris-HCl (pH 8.0), 1.5 mM [-P]ATP, and enzyme in 50 l for 30 min. at 37C.
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 fromE. coli lysogen NM989.
Performance and Quality Testing:Endodeoxyribonuclease, 3 and 5 exodeoxyribonuclease assays; ligation efficiency tested.
Unit Definition:One unit catalyzes the exchange of 1 nmol32P-labeled pyrophosphate into ATP in 20 min. at 37C. (One unit is equal to approximately 300 cohesive-end ligation units.)
Reduce the number of mutations in your synthetic gene or fragment
Reduce your labor time by screening only 24 clones instead of 10-16 clones per synthetic construct
Reduce your costs by sequencing only 24 clones instead of 1016 synthetic genes
Commercially available synthetic oligonuceotides have a high error rate during synthesis, ranging from one per 3001000 bases, depending on the source. These errors cause frameshift (deletion and insertion) and mismatch mutations during gene synthesis. Incubation with CorrectASE enzyme removes both type of mutations.
The incubation step with CorrectASE enzyme is introduced after the initial PCR assembly of oligonucleotides. The PCR product is denatured and reannealed so that any mutations will be unmatched. CorrectASE enzyme binds to the resulting mismatches and nicks both DNA strands 3 of the error. The 3to 5 exonuclease activity of the enzyme removes the errors. A final PCR with a proofreading polymerase then assembles the corrected fragments, thus increasing the likelihood of isolating clones with the correct sequence. Depending upon the incoming oligonuceotide quality, only 24 clones need to be screened, compared to 1016 clones in a workflow that does not include the correction step. Including CorrectASE enzyme in your gene synthesis workflow decreases labor time and sequencing costs.
For Research Use Only. Not for animal or human therapeutic or diagnostic use.
Exonuclease I, standard concentration, (10 units/L)
Exonuclease I hydrolyzes single-stranded DNA in the 35 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 theUSB ExoSAP-IT protocol. The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP.
Heat Inactivation: 80C for 15 min.
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating endonucleases, double-stranded exonucleases, and ribonucleases.
20mM Tris-HCI (pH 7.5), 5mM 2-mercaptoethanol, 0.5mM EDTA, 50% glycerol.
The reaction mixture (100 L) contains 67mM glycine buffer (pH 9.5), 10 mM
2-mercaptoethanol, 6.7 mM MgCl2, 0.5 mM denatured DNA, and enzyme. Incubation is at 37C for 30 min.
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 37C under standard conditions.
Standard Conc.: 10 units/L, PN 70073
High Conc.: 20 units/L, PN 72073
Functional Assay:Treated PCR product with Exonuclease I to degrade unincorporated primers before performing sequencing reaction with SequenaseVersion 2.0 DNA Polymerase Sequencing Kit (PN 70170).
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., VLKER, M. AND FIEHN, W. (1994)Nucleic Acids Res.22, 4354-4355.
Terminal Deoxynucleotidyl Transferase (20 U/L)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.
Incorporates modified nucleotides(e.g., fluorescein-, biotin-, aminoallyl-labeled nucleotides)
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 situlocalization of apoptosis
the TdT Reaction Buffer is incompatible with downstream applications. It is necessary to remove CoClfrom the reaction mixture by spin column or phenol/chloroform extraction and subsequent ethanol precipitation.
RNase A, DNase and protease-free (10 mg/mL)Thermo Scientific RNase A, DNase and protease-free is an endoribonuclease that specifically degrades single-stranded RNA at C and U residues.
It cleaves the phosphodiester bond between the 5-ribose of a nucleotide and the phosphate group attached to the 3-ribose of an adjacent pyrimidine nucleotide. The resulting 2, 3-cyclic phosphate is hydrolyzed to the corresponding 3-nucleoside phosphate.
RNase A is free of DNase activity. It is not necessary to heat it before use.
Plasmid and genomic DNA preparation
Removal of RNA from recombinant protein preparations
Ribonuclease protection assays. Used in conjunction with RNase T1
Mapping single-base mutations in DNA or RNA
Recommended concentration of RNase A is 1 to 100 g/mL depending on the application. The enzyme is active under a wide range of reaction conditions. At low salt concentrations (0 to 100 mM NaCl), RNase A cleaves single-stranded and double-stranded RNA as well the RNA strand in RNA-DNA hybrids. However, at NaCl concentrations of 0.3 M or higher, RNase A specifically cleaves single-stranded RNA.
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