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Enzyme Catalog Your trusted source for specialty molecular biology enzymes EPICENTRE Profile Introduction Our expertise in producing enzymes Unit definition Animal Product Free EPICENTRE Biotechnologies develops, manufactures, and sells high-quality enzyme systems for life science research. Located in Madison, Wisconsin, EPICENTRE was founded in 1987, and now occupies a state-of-the art 72,000-s.f. building. EPICENTRE is well-known for its unique expertise in making a broad range of enzymes for molecular biology, many of which are unique to EPICENTRE. EPICENTRE products are available directly in the United States and internationally through authorized distributors A significant number of commercially available enzymes have different unit definitions from different suppliers. Unit definition is directly related to the protocol on how to use an enzyme and to its actual cost. Some of our enzymes’ unit definition makes one EPICENTRE unt equivalent to multiple units of the same enzymes from some other suppliers. Please visit www.EpiBio. com or call our Tech Support at 1-800-2848474 if you have questions on our enzyme unit definition. We also produce a range of animal productfreeenzymes. Please visit www.EpiBio.com or call Customer Service at 1-800-274-8474 for more information Highest enzyme purity in the market EPICENTRE’s optimal and proprietary enzyme production procedure ensures that no tags are used in the production of our final enzyme products. If you are concerned about various tags affecting your research applications, EPICENTRE is your choice of enzyme source. EPICENTRE takes pride in its optimized and proprietary protein production, purification and quality control procedures that contribute to the extremely high purity of our enzymes products. Tag free Bulk and custom offerings EPICENTRE always tries to accommodate our customers’ needs for your specific applications. If you need enzymes with bulk quantitities, and/or custom formulations (e.g. higher concentrations), please inquire with our Customer Service at 1-800-274-8474. Limited use license EPICENTRE’s products are labeled for research or laboratory use only. For any orther use please inquire through our toll free number 1-800-2848474. NOTICE TO PURCHASER: LIMITED LICENSE *Use of MasterAmp™ AmpliTherm™ DNA Polymerase, MasterAmp™ Taq DNA Polymerase, MasterAmp™ Tfl DNA Polymerase, or MasterAmp™ Tth DNA Polymerase is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as the patented 5´ Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Use of MasterAmp™ PCR Enhancer DNA Polymerase Reactions, including, but not limited to use for PCR or DNA Sequencing, is covered by U.S. Patent No. 6,270,962, European Patent No. 0742838, German Patent No. DE4411588C1, and other issued or pending applications in the U.S. and other countries that are either assigned or exclusively licensed to EPICENTRE. These products are accompanied by a limited non-exclusive license for the purchaser to use the purchased products solely for life science research. Contact EPICENTRE for information on licenses for uses in diagnostics or other fields. 2 [email protected] • www.EpiBio.com T7, T3, and SP6 RNA Polymerases • Extremely high promoter specificity. Applications •RNA synthesized can be used as a hybridization probe, anti-sense RNA, a ribozyme, a template for in vitro translation, as a precursor mRNA for splicing or other processing studies, or to make dsRNA for RNA interference or gene silencing. •Synthesis of RNA for nucleic acid amplification methods or gene expression studies. Catalog No. Conc. T7 RNA Polymerase T7905K 50 U/µl T7925K 50 U/µl TM910K 200 U/µl TM925K 200 U/µl TH950K 1,000 U/µl TU950K 2,500 U/µl Size Price $ 5,000 U 25,000 U 10,000 U 25,000 U 50,000 U 50,000 U (Enzyme only. Transcription Buffer is not included.) T3 RNA Polymerase T4905K 50 U/µl T9050K 50 U/µl TM4910K 200 U/µl TH4950K 1,000 U/µl 5,000 U 50,000 U 10,000 U 50,000 U (Enzyme only. Transcription Buffer is not included.) SP6 RNA Polymerase S4905K 50 U/µl S4950K 50 U/µl SM910K 200 U/µl 5,000 U 50,000 U 10,000 U (Enzyme only. Transcription Buffer is not included.) RNA Polymerases and Replicases Produce defined RNA by in vitro transcription of double-stranded DNA that is downstream of the respective RNA polymerase promoter. Transcription Buffer Package BP1001 1 pkg Includes 5 ml of 5X Transcription Buffer and 2.5 ml of 100 mM DTT. *Greatest range of enzyme concentrations available from 50 U/µl to 2,500 U/µl. **For kits for in vitro transcription, please visit www.EpiBio.com/ivt.asp. T7 R&DNA™ Polymerase and SP6 R&DNA™ Polymerase Recombinant enzymes having single-base active-site mutations in the respective T7 or SP6 RNA Polymerase gene. These active-site mutations enable the corresponding T7 or SP6 R&DNA Polymerase to incorporate 2’deoxyribonucleoside triphosphates (dNTPs) into full-length transcripts much more efficiently than the corresponding wild-type enzymes, while retaining the same catalytic activity for incorporation of canonical NTPs and the same high promoter specificity. Applications •Synthesis of “RNA” transcripts of mixed rNMP/2’-dNMP or rNMP/2’-modified-NMP composition. •Synthesis of modified “RNA” transcripts that are resistant to RNase A-type RNases. T7 & SP6 R&DNA™ Polymerases, & DuraScribe™ T7 & SP6 Transcription Kits to synthesize nucleic acids with non-canonical bases or for partial ribosubstitution are covered by U.S. Patents 5,849,546; 6,107,037; or 6,596,494, and other patents issued or pending. These products are accompanied by a limited nonexclusive license for the purchaser to use the purchased product(s) solely for life science research except the development of therapeutics. Contact EPICENTRE concerning licenses for other uses. Catalog No. Conc. T7 R&DNA™ Polymerase D7P9201K 50 U/µl D7P9205K 50 U/µl Size Price $ 1,000 U 5,000 U Contents: T7 R&DNA™ Polymerase, 5X Reaction Buffer, and 100 mM DTT. SP6 R&DNA™ Polymerase D6P9301K 50 U/µl D6P9305K 50 U/µl 1,000 U 5,000 U Contents: SP6 R&DNA™ Polymerase, 5X Reaction Buffer, and 100 mM DTT. *For kits for in vitro transcription, please visit www.EpiBio.com/ivt.asp. E. coli RNA Polymerase Core Enzyme and Sigma-Saturated Holoenzyme Both enzyme preparations are isolated from the rifampicin-sensitive strain BL21. EPICENTRE is the only company that offers purified Core Enzyme, which has no detectable sigma subunit, and 100% Sigma-Saturated (σ70)-Holoenzyme. Applications •The Core Enzyme is useful in studying mechanisms of transcription initiation, since it will not initiate specific transcription at promoter sequences on bacterial or bacteriophage DNA due to a lack of sigma factor. •The sigma-saturated Holoenzyme is very efficient in specifically transcribing a variety of double-stranded DNA templates containing promoters. Holo Core - β´, β -σ -α -ω FIG 1. Subunit patterns of E. coli RNA Polymerase Core Enzyme and Holoenzyme preparations. Equivalent amounts of each enzyme were separated by electrophoresis on an SDS 15% polyacrylamide gel, stained with Coomassie Blue, and dried. The sigma-70 subunit is 100% saturating in the Holoenzyme, but is absent in the Core Enzyme. Catalog No. Conc. Size E. coli RNA Polymerase Core Enzyme C90100 100 U C90250 250 U E. coli RNA Polymerase Holoenzyme (Sigma-Saturated) S90050 50 U S90100 100 U *For kits for in vitro transcription, please visit www.EpiBio.com/ivt.asp. Thermus Thermostable RNA Polymerase Derived from the thermophilic bacterium, Thermus thermophilus, this RNA Polymerase is the only commercially-available RNA www.EpiBio.com • [email protected] polymerase that is stable and has optimal activity at temperatures above 65°C. Catalog No. Conc. Size Price $ Thermus Thermostable RNA Polymerase T90050 50 U T90100 100 U 3 RNA Capping and Tailing Enzymes ScriptCap™ m7G Capping System Catalog No. Conc. Size ScriptCap™ m7G Capping System SCCE0610 - 10 Reactions SCCE0625 - 25 Reactions Contents: ScriptCap™ Capping Enzyme, 10X Capping Buffer, ScriptGuard™ RNase Inhibitor, 20 mM SAM, 10 mM GTP, and RNase-Free Water. *For complete kits designed to produce capped and tailed RNA in vitro, please visit www.EpiBio.com/capping.asp. Based upon the tri-functional Vaccinia Virus capping enzyme (VCE), this system is designed to build the Cap 0 structure found on the 5´ end of most eukaryotic mRNA molecules. This enzyme system is identical to the Vaccinia guanylyltransferase sold by other vendors. Applications • In vitro production of capped RNA for in vivo or in vitro translation.* •Analysis of 5´ ends of RNA transcripts. Figure: (A) Denaturing Polyacrylamide gel and (B) autoradiograph of a ScriptCap™ Capping system reaction. Lane 2 shows the uncapped transcript without Vaccinia capping enzyme (VCE). Lane 3 includes the VCE and 14C SAM. VCE has clearly transferred the guanine base (Lane 3A) and the 14C containing methyl group (Lane 3B). ScriptCap™ 2’-O-Methyltransferase Catalog No. Conc. Size ScriptCap™ 2’-O-Methyltransferase SCMT0610 - 10 Reactions SCMT0625 - 25 Reactions Contents: ScriptCap™ 2’-O-Methyltransferase, 10X Capping Buffer, and 20 mM SAM. * For complete kits designed to produce capped and tailed RNA in vitro, please visit www.EpiBio.com/capping.asp. The ScriptCap 2’-O-Methyltransferase Enzyme is derived from the Vaccinia virus and methylates the penultimate nucleotide in a capped eukaryotic mRNA transcript, converting a Cap 0 transcript into the natural Cap 1 mRNA structure. This methylation results in an up to 50% increase in the in vivo translation efficiency when compared to Cap 0 mRNA. The enzyme is active on both the natural Cap 0 structure and several different Cap 0 dinucleotide analogs used in co-transcriptional capping kits. Each reaction is capable of methylating 60 ug of Cap 0 RNA in 30 minutes. Applications Table 1. Various different forms of capped and tailed mRNA were transfected into HeLa cells and assayed for luciferase activity. Data is normalized to the Cap 0, poly(A) transfection results. The complete Cap 1 mRNA exhibited up to 50% higher in vivo translation efficiency when compared to the various Cap 0-type structures currently used in most transfections. Methylation of Cap 0 RNA to the Cap 1 form Means of Cap 0 Production ScriptCap™ Final mRNA Cap 2’-O-Methyltransferase Structures Formed Treatment No RNA no none ScriptCap™ m7G Capping System no m7GpppN (Capping Enzyme) (Cap 0) ScriptCap™ m7G Capping System yes m7Gppp[m2’-O]N (Capping Enzyme) (Cap 1) AmpliCap-Max™ High Yield no m7GpppN * Message Maker Kit (Cap 0) (Standard Cap Analog) AmpliCap-Max™ High Yield yes m7Gppp[m2’-O]N * Message Maker Kit (Cap 1) (Standard Cap Analog) AmpliScribe™ T7-Flash™ no m27, 3´-OGpppN * Transcription Kit (Cap 0) (ARCA Cap Analog) AmpliScribe™ T7-Flash™ yes m27, 3´-OGppp[m2’-O]N * Transcription Kit (Cap 1) (ARCA Cap Analog) Translation Efficiency relative to non-treated mRNA 0% 100% 147% 100% 148% 100% 128% A-Plus™ Poly(A) Polymerase Catalog No. Conc. Size A-Plus™ Poly(A) Polymerase Tailing Kit 50 PAP5104H 4 U/µl Reactions (400 U) Contents: A-Plus™ Poly(A) Polymerase, A-Plus™ 10X Reaction Buffer, 10 mM ATP, and Sterile RNase-Free Water. A-Plus™ Poly(A) Polymerase uses ATP as a substrate for template-independent addition of adenosine monophosphate to the 3´-hydroxyl termini of RNA molecules. The A-Plus™ Poly(A) Polymerase Tailing Kit provides the enzyme and other reagents to quickly and easily add a “poly(A) tail” to the 3´-end of any RNA. Applications •Addition of a poly(A) tail to RNA synthesized in vitro to increase RNA stability and enhance its in vivo translation after transfection or microinjection into eukaryotic cells. • Addition of a poly(A) tail to RNA molecules to 4 provide a priming site for synthesis of firststrand cDNA using a primer with poly(dT) on its 3´-end. •Cloning of DNA encoding RNA molecules of unknown or multiple sequences by adding a poly(A) tail that can anneal to a T-tailed vector. •Synthesis of polyadenylated RNA for nucleic acid amplification methods or gene expression studies. •3´-End-labeling of RNA with radioactive A residues. •Quantifying mRNA. [email protected] • www.EpiBio.com Properties of Mesophilic DNA Polymerases 3´→5´ exonuclease Activity Nick translation Heat Inactivationa Strand displacement DNA Polymerase I, E. coli + ++ + 75°C 20 minutes - Klenow DNA Polymerase - ++ - 75°C 20 minutes + Exo-Minus Klenow DNA Polymerase - - - 75°C 20 minutes + RepliPHI™ Phi29 DNA Polymerase - ++ - 65°C 10 minutes ++++ T4 DNA Polymerase - +++ - 75°C 20 minutes - T7 DNA Polymerase, Unmodified - +++ - 75°C 20 minutes - a DNA Polymerases 5´→3´ exonuclease Product Name Indicated treatment results in complete inactivation under standard reaction conditions DNA Polymerase I, E. coli This DNA-dependent DNA polymerase contains both 5’→3’and 3´→5´ exonuclease activities. The 5´→3´ exonuclease activity enables the enzyme to use nicks and gaps in the DNA as starting points for labeling the DNA by nick translation. Applications •Generate labeled DNA probes by nick translation. •Second strand cDNA synthesis Catalog No. Conc. DNA Polymerase I, E. coli DP02250 10 U/µl DP021K 10 U/µl Size 250 U 1000 U • In vitro synthesis of DNA. Klenow DNA Polymerase Derived from E. coli DNA polymerase I, this large fragment, DNA-dependent enzyme has 5´→3´ polymerization and 3´→5´ exonuclease activities, but lacks 5´→3´ exonuclease activity. Klenow DNA polymerase blunt ends doublestranded DNA with singlestranded overhangs. The 3´→5´ exonuclease activity removes 3´ overhangs and the 5´→3´ polymerization activity fills in 5´ overhangs. Applications • Random primer labeling of DNA. • DNA sequencing. Catalog No. Conc. Klenow DNA Polymerase KP04061K - Size 1000 U • Second-strand cDNA synthesis. • Strand displacement amplification. Exo-Minus Klenow DNA Polymerase This DNA-dependent DNA polymerase lacks both the 5´→ 3´ and 3´→5´ exonuclease activities of E. coli DNA Polymerase I from which it is derived. Applications • Random primer labeling of DNA. • DNA sequencing. Catalog No. Conc. Size Exo-Minus Klenow DNA Polymerase KL04011K 20 U/µl 1000 U KL06041K 50 U/µl 1000 U • Second-strand cDNA synthesis. • Strand displacement amplification. RepliPHI™ Phi29 DNA Polymerase RepliPHI™ Phi29 DNA Polymerase (φ29 DNA Polymerase) is a highly processive enzyme with exceptional strand displacement activity. The www.EpiBio.com • [email protected] enzyme also contains a 3´→5´ exonuclease activity that enables proofreading capability. Its specific activity is 1 x 106 U/mg. Catalog No. Conc. Size RepliPHI™ Phi29 DNA Polymerase (enzyme only) 10 µg 1 µg/µl PP031010 (1,000 U/µl) (10,000 U) 10 µg 0.1 µg/µl PP040110 (100 U/µl) (10,000 U) RepliPHI™ Phi29 Reagent Set (includes enzyme, buffer, dNTPs, DTT) Enzyme: Enzyme: RH031110 1 µg/µl 10 µg (1,000 U/µl) (10,000 U) Enzyme: Enzyme: RH040210 0.1 µg/µl 10 µg (100 U/µl) (10,000 U) RepliPHI™ Phi29 Polymerase Dilution Buffer RPB04041 1 ml 5 DNA Polymerases T4 DNA Polymerase Catalog No. Conc. T4 DNA Polymerase D0602H D0605H - Contains both a template-directed 5´→3´ DNA polymerase activity and a potent 3´→ 5´ exonuclease activity. Size 200 U 500 U Applications •Conversion of 5´- and 3´-protruding DNA termini to blunt ends. •Cloning of PCR fragments: Treatment of PCR products containing 3´-A overhangs with T4 DNA Polymerase and dNTPs produces blunt ends. •Production of site-specific mutations: this enzyme can be used for site-specific mutagenesis by primer extension of “mutated” oligonucleotides hybridized to single-stranded DNA templates. •Labeling of 3´-termini of DNA molecules and synthesis of strand-specific probes using the exonuclease and polymerase activities of T4 DNA Polymerase. T7 DNA Polymerase, Unmodified Contains both a template-directed 5´→ 3´ DNA polymerase activity and a potent 3´→ 5´ exonuclease activity. The unmodified form of T7 DNA Polymerase is different from T7 DNA polymerase preparations from which exonuclease activity has been removed. The enzyme is a tightly-bound complex of T7 phage-encoded gene 5 protein and E. coli hostencoded thioredoxin. Catalog No. Conc. Size T7 DNA Polymerase, Unmodified D07250 10 U/µl 250 U D07500 10 U/µl 500 U Highly processive and synthesizes long stretches of DNA before dissociating from the template. The rate of elongation is also much faster than that of most other DNA polymerases. Applications •Primer extension of long DNA molecules. •Conversion of 5´- and 3´-protruding ends to blunt ends. •Labeling of 3´-ends of DNA. • In situ detection of DNA fragmentation associated with apoptosis. Properties of Thermophilic DNA Polymerases Product Name Activity Thermostabilityb Fidelityc Strand displacement <15 min at 75°C <1.5 min at 95°C N/A - - <15 min at 75°C <1.5 min at 95°C N/A +++ - - N/A N/A N/A Very weak. Requires Mn2+ + - 9 min at 97.5°C 0.38-1.82 x 104 N/A MasterAmp™ Tfl DNA Polymerase - + - 40 min at 95°C 8.3-9.0 x 105 N/A MasterAmp™ Tth DNA Polymerase ++ Requires Mn2+ + - 20 min at 95°C 2.2 x 104 N/A Reverse transcriptase 5´→3´ exonuclease 3´→5´ exonuclease rBst DNA Polymerasea + + - rBst DNA Polymerase Large Fragment, (IsoTherm™ DNA Polymerase)a - - MasterAmp™ AmpliTherm™ DNA Polymerase - MasterAmp™ Taq DNA Polymerase a rBst DNA polymerase and rBst DNA Large Fragment (IsoTherm™ DNA Polymerase) are for DNA replication at < 65°C. Not suitable for typical PCR. The other thermostable enzymes listed in this chart are suitable for PCR cycling applications. b Values represent half-lives: 50% of the enzymatic activity is retained after the given time at the stated temperature. c Defined as the average number of correct nucleotides a polymerase incorporates before making an error. rBst DNA Polymerase Catalog No. Conc. rBst DNA Polymerase BH1100 5 U/µl BH1500 5 U/µl Size 100 U 500 U Derived from the DNA pol I gene of the thermophilic bacterium Bacillus stearothermophilus (Bst), this enzyme has optimal activity at 65°C and at elevated temperatures it can synthesize DNA in regions containing template secondary structure or high GC content where other non-thermostable DNA polymerases may fail. It has 5´→ 3´ exonuclease activity. Its optimal activity as elevated temperature 6 makes it useful for replicating difficult templates. Applications •High temperature DNA synthesis. •First strand cDNA synthesis from primed RNA templates. •Production of cDNA templates for use in amplification reactions. [email protected] • www.EpiBio.com rBst DNA Polymerase, Large Fragment (IsoTherm™ DNA Polymerase) Its high rate of DNA synthesis permits use of nanogram quantities of template under isothermal conditions. Ability to synthesize through regions of high GC content or difficult secondary structure at high temperatures. •High-temperature isothermal DNA sequencing. Applications •Amplification assays and other assays involving continuous displacement of the DNA strand concomitant with high-temperature DNA synthesis. Catalog No. Conc. Size rBst DNA Polymerase, Large Fragment (IsoTherm™ DNA Polymerase) BL901K 5 U/µl 1,000 U BL1805K 50 U/µl 5,000 U BL1950K 50 U/µl 50,000 U Enzyme only. •First-strand cDNA synthesis from primed RNA templates. DNA Polymerases Derived from the DNA pol I gene of the thermophilic bacterium Bacillus stearothermophilus (Bst) altered to remove the 5´→ 3´ DNA exonuclease activity. Having optimal DNA polymerase activity at 65°C, the enzyme is suitable for high-temperature isothermal sequencing of DNA. Like the Klenow fragment of E. coli DNA Polymerase I, this enzyme has strong strand displacement activity. It also has thermostable RNA-dependent DNA polymerase (i.e., reverse transcriptase) activity. MasterAmp™ AmpliTherm™ DNA Polymerase* This proprietary recombinant thermostable DNA polymerase for use in PCR has optimal DNA synthetic activity at temperatures above 70°C and can be used at temperatures up to 95°C. It lacks both 5´→ 3´ structure-dependent exonuclease activity like that found in Taq DNA polymerase and 3´→ 5´ proofreading exo- nuclease activity found in some other DNA polymerases. Provided with the MasterAmp™ PCR Enhancer, which increases the probability of obtaining the desired amplification product, the reproducibility of PCR, and improves the consistency of PCR product yields in multiplex PCR. Applications • PCR amplification of DNA templates. • Multiplex PCR. Catalog No. Conc. Size MasterAmp™ AmpliTherm™ DNA Polymerase AT72250 5 U/µl 250 U Includes 10X PCR Buffer, 25 mM MgCl2, and MasterAmp™ 10X PCR Enhancer. MasterAmp™ AmpliTherm™ DNA Polymerase (Enzyme Only) AT72250N 5 U/µl 250 U For use with MasterAmp™ PCR PreMixes. MasterAmp™ Taq DNA Polymerase* This thermostable DNA polymerase derived from Thermus aquaticus has optimal activity at temperatures above 70°C. It has an intrinsic 5´→ 3´ structure-dependent exonuclease activity, but lacks 3´→ 5´ proofreading exonuclease activity. Reliable activity, specificity, and reproducibility in PCR in conjunction with the MasterAmp PCR Enhancer Technology. Applications •PCR and Multiplex PCR amplification of DNA templates. Catalog No. Conc. Size MasterAmp™ Taq DNA Polymerase Q82250 5 U/µl 250 U Q8201K 5 U/µl 1,000 U Includes 10X PCR Buffer, 25 mM MgCl2, and MasterAmp™ 10X PCR Enhancer. MasterAmp™ Taq DNA Polymerase (Enzyme Only) Q82250N 5 U/µl 250 U For use with MasterAmp™ PCR PreMixes. MasterAmp™ Taq PCR Core Kit 200 Reactions MCQ74200 Contents: MasterAmp™ Taq DNA Polymerase, 10X PCR Buffer, MasterAmp™ 10X PCR Enhancer, dNTP Mix, 2.5 mM each, 25 mM MgCl2, Enzyme Dilution Buffer, Control Template and Primers Mix. MasterAmp™ Tfl DNA Polymerase* Derived from the thermophilic bacterium Thermus flavus, this is a recombinant DNA polymerase with good thermostability (to ~95°C) and processivity (with 15 kb PCR products reported). MasterAmp™ PCR Enhancer Technology substantially increases the probability of obtaining the desired amplification product and the reaction-to-reaction consistency, and greatly improves the consistency of PCR product yields in multiplex PCR. Applications •PCR and Multiplex PCR amplification of DNA templates. •Produces PCR products up to 15 kb. •Adaptable to high-throughput PCR formats. Catalog No. Conc. Size MasterAmp™ Tfl DNA Polymerase F72250 1 U/µl 250 U F7201K 1 U/µl 1,000 U Includes 20X PCR Buffer, 25 mM MgCl2, and MasterAmp™ 10X PCR Enhancer. MasterAmp™ Tfl DNA Polymerase (Enzyme Only) F72250N 1 U/µl 250 U For use with MasterAmp™ PCR PreMixes. MasterAmp™ Tth DNA Polymerase* This recombinant DNA enzyme from Thermus thermophilus has both DNA-dependent and RNA-dependent (i.e., reverse transcriptase) DNA polymerase activities up to ~95°C. High reaction temperatures can reduce nonspecific priming and template secondary structure. Provided with MasterAmp™ PCR Enhancer Technology. dependent DNA polymerase activity under the same reaction conditions. Has both reverse transcriptase and DNA- •1-step RT-PCR of RNA templates. Improved PCR of RNA and DNA templates having a high degree of secondary structure. Applications •PCR amplification of DNA Catalog No. Conc. Size MasterAmp™ Tth DNA Polymerase TTH72100 5 U/µl 100 U TTH72250 5 U/µl 250 U TTH7201K 5 U/µl 1,000 U Includes a 20X PCR Buffer (without Mg2+ or Mn2+) plus separate 25 mM solutions of MgCl2 and MnSO4, and MasterAmp™ 10X PCR Enhancer. MasterAmp™ Tth DNA Polymerase (Enzyme Only) TTH7225N 5 U/µl 250 U For use with MasterAmp™ PCR PreMixes. www.EpiBio.com • [email protected] *Refer to page 2 for patent and license information. 7 Catalog No. Conc. Size MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit MM070110 10 Reactions MM070150 50 Reactions Contents: MMLV Reverse Transcriptase, 10X Reaction Buffer, ScriptGuard™ RNase Inhibitor, dNTP PreMix, DTT, Oligo(dT)21Primer, Random 9-mer Primers, RNase-free Water. *See page XX for RNase H and related products The MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit is optimized for generating full-length first-strand cDNA from total cellular RNA preparations or purified polyadenylated RNA. •Synthesize full-length cDNA from RNA templates longer than 15 kb. ~ 15.2 kb HERC1 mRNA 5' 1A AAAA(A)n 3' cDNA Synthesis 3' TTTT(T)17 5' 1.3 kb PCR product •Both oligo(dT) and random primers included in the kit. i2130703 Reverse Transcriptase MMLV Reverse Transcriptase 1B •The kit includes both an oligo(dT)-containing and a random nonamer (9-mer) primer. •First-strand cDNA can be made from picogram amounts of total RNA. • A potent RNase Inhibitor is included. Applications •First-strand cDNA synthesis. •Production of cDNA for subsequent PCR or real-time PCR. •RT-PCR validation of gene expression data obtained from microarray experiments. •RT-PCR validation and quantification of gene silencing by RNA interference. FIG 1. The MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit produces full-length cDNA from mRNA longer than 15 kb. Total RNA isolated from HeLa cells was reverse transcribed and the cDNA was amplified as described in the text. A. Detection of the 1.3 kb PCR amplicon from near the 5´ end of the mRNA demonstrates full-length reverse transcription of HERC1 mRNA. B. Agarose gel analysis of the PCR reaction shows the 1.3 kb amplicon from the 5´-end of the mRNA. Lane M, 100 bp DNA ladder; Lane 1, no reverse transcriptase control reaction; Lane 2, PCR product from cDNA synthesized by EPICENTRE’s MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit. MonsterScript™ 1st-Strand cDNA Synthesis Kit The MonsterScript™ 1st-Strand cDNA Synthesis Kit is optimized for generating full-length first-strand cDNA from multiple mRNA species in samples containing total cellular or purified polyadenylated RNA. This kit uses MonsterScript™ Reverse Transcriptase, a reverse transcriptase that lacks RNase H activity. The enzyme’s lack of RNase H activity contributes to its ability to make longer cDNAs and more complete libraries of first-strand cDNA molecules. Applications •Lacks RNase H, enabling improved synthesis of full-length cDNA even for long mRNA. 5' •MonsterScript is thermostable, permitting reverse transcription at temperatures >50°C, which reduces RNA secondary structure and improves priming specificity. •MonsterScript™ RT PreMix contains optimized concentrations of dNTPS, Mg+2 and Betaine for superior performance and minimal pipetting steps. •Betaine in the cDNA Synthesis PreMix reduces pausing and stops during reverse transcription. •The kit includes both an oligo(dT)-containing and a random nonamer primer. •First-strand cDNA can be made from picogram amounts of total RNA. •A potent RNase Inhibitor is included. 8 • First-strand cDNA synthesis. •Production of cDNA for subsequent PCR or real-time PCR. •RT-PCR validation of gene expression data obtained from microarray experiments. •RT-PCR validation and quantification of gene silencing by RNA interference. ~ 15.2 kb HGNEFp532 mRNA 1A AAAAA . . . -3' i470407ms Catalog No. Conc. Size MonsterScript™ Reverse Transcriptase MSTA5110 10 Reactions MSTA5124 24 Reactions 1.3 kb PCR 1B —1.3 kb FIG 1. MonsterScript™ Reverse Transcriptase produces full-length cDNA from mRNA greater than 15 kb. The ≈15.2 kb HGNEFp532 mRNA was reverse transcribed from total HeLa RNA in a standard MonsterScript reaction. Two microliters of the reaction was used to PCR amplify a 1.3 kb region within 68 bases of the 5´-end of the HGNEFp532 mRNA (1A). Agarose gel of the 1.3 kb amplicon from the 5´-end of the mRNA demonstrates full-length cDNA synthesis (1B). [email protected] • www.EpiBio.com Properties of Exonucleases and Endonucleases: Active on both DNA and RNA Enzyme Terminator™ 5´-PhosphateDependent Exonuclease Substrate Activity Products Applications Heat Inactivate ssDNA or ssRNA 5´→ 3´ exonuclease that digests ssDNA or ssRNA with 5´-phosphorylated ends, but not with 5´-hydroxylated ends. dNMPs or NMPs Removal of 5’-phosphorylated DNA or primers or oligos. Enrichment of ssDNA or ssRNA molecules lacking 5’-phosphate groups. 65°C for 10 min Single-strand-specific endonuclease. dNMPs or NMPs and oligos with 5´-phosphate and 3´-OH Converting 5´ or 3´ overhangs to blunt ends. Removing hairpins after cDNA synthesis. Detecting SNPs by digesting single-base mismatches. With exo III, for making nested deletions.“S1-type” mapping of RNA. No Endonuclease that efficiently digests DNA and RNA. di-, tri-, and tetra-nucleotides Removal of DNA and RNA from protein preps. Removal of host DNA from phage preps. No Nucleases DNA and RNA Exonuclease DNA and RNA Endonucleases Mung Bean Nuclease OmniCleave™ Endonuclease ssDNA or ssRNA single- and double-stranded DNA and RNA Terminator™ 5´-Phosphate-Dependent Exonuclease Terminator Exonuclease is a 5´-to-3´, processive exonuclease that degrades RNAs that have a 5´-monophosphate. RNAs with a 5-triphoaphate, 5´-cap structure such as found on most eukaryotic mRNAs or a 5´-OH are resistant to Terminator Exonuclease degradation. It will also digest DNA with a 5´-monophosphate. It is not inhibited by proteinaceous RNase inhibitors. Applications •Characterize the 5-terminii of RNA transcripts. •Prepare mRNA-enriched samples from eukaryotic or prokaryotic total RNA preparations in 1 hour without the use of Oligo(dT), resins or magnetic beads. Catalog No. Conc. Size Terminator™ 5´-Phosphate-Dependent Exonuclease TER51020 1 U/µl 20 U *Patent Pending. Terminator™ Exonuclease 1 Hour Large rRNA mRNA FIG 2. A 1-hour Terminator™ Exonuclease reaction digests the large rRNAs in a eukaryotic or prokaryotic total RNA sample, producing an enriched-mRNA preparation. FIG 3. Denaturing agarose gel analysis of E. coli total RNA before (-) and after (+) Terminator™ Exonuclease digestion. The Terminator Exonucleasetreated RNA was concentrated 10-fold. FIG 4. Normal rat kidney (NRK) total RNA before (A) and after (B) Terminator™ Exonuclease treatment. The Terminator Exonuclease-treated RNA was concentrated 10-fold. www.EpiBio.com • [email protected] 9 Nucleases Mung Bean Nuclease Catalog No. Conc. Mung Bean Nuclease M8202K 50 U/µl M8205K 50 U/µl This single-strand-specific nuclease has higher specificity for single-stranded DNA and RNA than S1 Nuclease. Unlike S1 Nuclease, Mung Bean Nuclease will not cleave the intact strand of nicked duplex DNA. Size 2,000 U 5,000 U Applications •High resolution mapping of the termini and exon structures of RNA transcripts (commonly termed Berk-Sharp or S1-Mapping) using either internal-labeled or end-labeled probes. •Restriction site modification or removal by digestion of single-stranded protruding ends. •Removal of hairpin structures during cDNA synthesis. •Cleavage of single-basepair mismatches. Digests all forms of DNA and RNA including single-stranded and double-stranded linear, circular, and supercoiled. OmniCleave Endonuclease has the same substrate specificity and yields the same products as Benzonase®, an enzyme derived from Serratia marcescens. Applications OmniCleave™ Endonuclease Catalog No. Conc. Size OmniCleave™ Endonuclease OC7810K 200 U/µl 10,000 U OC7850K 200 U/µl 50,000 U Provided with Dilution Buffer. •Improves handling and yield of protein preparations by reducing the viscosity of cell lysates due to nucleic acids. •Removes trace contamination by nucleic acids in protein preparations. •Removes host DNA from phage preparations. Plasmid-Safe™ ATP-Dependent DNase Catalog No. Conc. Size Plasmid-Safe™ ATP-Dependent DNase E3101K 10 U/µl 1,000 U E3105K 10 U/µl 5,000 U E3110K 10 U/µl 10,000 U Plasmid-Safe™ ATP-Dependent DNase selectively removes contaminating bacterial chromosomal DNA from cosmid, BAC, fosmid, and plasmid preparations. The enzyme will processively degrade linear DNA from the ends; closed circular DNA (i.e. a plasmid) does not have free ends, and is therefore not degraded. These properties make PlasmidSafe™ ATP-Dependent DNase ideal for BAC and fosmid purification protocols such as shot-gun sequencing and FISH where high purity DNA is a must. Applications •Removal of contaminating bacterial chromosomal DNA in large-scale plasmid, cosmid, fosmid, BAC or vector preparations. FIG 1. Plasmid-Safe™ ATPDependent DNase removes contaminating genomic DNA from plasmid preps. –, mixture of 3 µg of digested bacterial chromosomal DNA and 500 ng of uncut plasmid before Plasmid-Safe™ DNase treatment; +, mixture of chromosomal DNA and plasmid DNA after Plasmid-Safe™ DNase treatment (incubated with Plasmid-Safe™ DNase for 30 minutes at 37°C); M, kb ladder. Properties of DNA Endonucleases DNA Endonucleases Enzyme 10 Substrate Activity Products Applications Heat Inactivate Baseline-ZERO™ DNase dsDNA and ssDNA Digests dsDNA or ssDNA down to mononucleotides mononucleotides Removing DNA from RNA prep 75°C for 20 min DNase I (bovine pancreas) E.C. 3.1.21.1 dsDNA and ssDNA Activated by divalent cations. In presence of Mg2+, it cleaves each DNA strand randomly and independently, preferentially adjacent to pyrimidines. In presence of Mn2+, it cleaves both strands simultaneously, generating fragments with blunt ends or 1-2-base overhangs. Oligos and dNMPs with 5´-phosphate and 3´-OH. Removing DNA from RNA preps. Random nicking of dsDNA. DNase footprinting. 75°C for 20 min Endonuclease IV (E. coli) dsDNA with abasic site Cleaves sugar-phosphate bond 5´ of an abasic site. dsDNA with singlenucleotide gaps. The cleaved ssDNA strand has a 3´-OH. DNA repair and anti-tumor drug research. Base Excision Sequence Scanning of DNA containing dUMPs. N/A T4 Endonuclease V UV-irradiated DNA with thymine dimers First cleaves N-glycosidic bond 5´ of thymine dimers, then cleaves sugar-phosphate bond 3´ of the abasic site. Nicked dsDNA with an abasic site at the 3´-end of the cut and thyminedimer bases at the 5´-end of the cut. Research on repair of DNA exposed to UV light. N/A Lambda Terminase dsDNA with cos sites Cleaves both strands at bacteriophage lambda cos sites. 5´-ends with overhangs 12 bases in length. Rapid sizing or restriction mapping of BAC, fosmid or cosmid clones. N/A [email protected] • www.EpiBio.com Baseline-ZERO™ DNase Provides a true zero baseline for RNA RT-PCR or microarray gene expression experiments. Applications •Removal of genomic DNA from RNA prior to RT-PCR or preparation of target RNA or cDNA for microarray analysis, esp. for exon arrays or full coverage expression analysis •Elimination of the DNA template following in vitro RNA synthesis with T7, T3 or SP6 Phage RNA Polymerases. •Removal of ssDNA and dsDNA from viral RNA. •Elimination of genomic DNA from RNA for microinjection and transfection experiments. •As a replacement of DNase I in applications requiring the removal of all DNA contamination. •Reverse transcription of RNA using a random primer since any contaminating DNA would also be a template for random-primed cDNA synthesis. Catalog No. Conc. Baseline-ZERO™ DNase DB0711K 1 U/ul DB0715K 1 U/ul FIG. Demonstration of the use of BaselineZERO™ DNase to remove small oligonucleotides during DNase treatment. Lane 1, kilobase ladder; Lanes 2-5, 160 ng of EcoR I-digested plasmid DNA incubated for 15 minutes at 37°C as follows: Lane 2, untreated; Lane 3, incubated with DNase I: Lane 4, with supplier A’s hyper-active DNase; Lane 5, with Baseline-ZERO™ DNase. Only BaselineZERO™ DNase removes the small residual oligos at the bottom of the gel. *Patent Pending Size 1,000 MBU 5,000 MBU *Special introductory prices Nucleases Digests double- and single-stranded DNA to mononucleotides more effectively than the commonly used bovine pancreatic DNase I. Following treatment with Baseline-ZERO, even the small DNA oligonucleotides that remain after treatment with bovine pancreatic DNase I are undetectable. Oligos 1 2 3 4 5 RNase-Free DNase I RNase-Free DNase I (bovine pancreas) is an endonuclease useful in removing DNA that might interfere with the characterization, manipulation, or use of RNA, or for any application requiring highly purified DNase I. It efficiently hydrolyzes double- and single-stranded DNA to a mixture of short oligo- and mononucleotides. 1 2 3 —DNA •Labeling of DNA by nick translation, in combination with Klenow or other DNA polymerases. • Treatment of RNA prior to RT-PCR. •Characterization of DNA-protein interactions by DNase I footprinting. Catalog No. Conc. RNase-Free DNase I D9902K D9905K - Size 2,500 MBU 5,000 MBU Supplied at a concentration of 1 U/µl. —transcript Applications •Elimination of template DNA following in vitro synthesis of RNA with T7, SP6, or T3 phage RNA polymerase. 4 FIG 1. Complete DNA removal from in vitro transcription reactions using RNase-Free DNase I. A linearized DNA template was transcribed using T7 RNA polymerase according to standard in vitro transcription conditions. Lane 1, kb ladder; Lane 2, DNA control; Lane 3, transcription mixture; Lane 4, transcription mixture treated with 1 MBU of RNase-Free DNaseI for 15 minutes at 37°C. Endonuclease IV, E. coli Endonuclease IV, cloned from the E. coli nfo gene, is a metalloenzyme that functions in vivo to repair free radical damage in DNA. The enzyme also has Class II abasic endonuclease activity, which has utility in many areas of DNA damage and repair research. It is also useful in the study of the effects of anti-tumor drugs such as bleomycin on nucleic acids in vivo. Applications Catalog No. Conc. Endonuclease IV, E. coli E70100 2 U/µl Size 100 U •DNA repair research. •Anti-tumor drug evaluation. T4 Endonuclease V T4 Endonuclease V has N-glycosylase and apurinic/apyrimidinic lyase (AP lyase) activities. Ultraviolet (UV) light produces covalent photoproducts in DNA, the most prevalent being a cis-syn cyclobutane pyrimidine dimer. T4 Endonuclease V locates and binds to pyrimidine dimers in double-stranded DNA, then cleaves the N-glycosylic bond of the 5´-pyrimidine of the dimer (pyrimidine dimer DNA glycosylase) www.EpiBio.com • [email protected] and breaks the phosphodiester bond 3´ to the resulting abasic site (3´ AP lyase). Applications •Study of UV damage to DNA and its repair, including DNA damage in single cells. Catalog No. Conc. T4 Endonuclease V TE6605 20 U/µl TE661K 20 U/µl TE665K 20 U/µl Size 500 U 1,000 U 5,000 U •Detection of differential UV damage repair of transcribed sequences. •Detection of UV mutational hotspots. 11 Nucleases Lambda Terminase Catalog No. Conc. Lambda Terminase LT4450 2 U/µl LT44200 2 U/µl Size 50 U 200 U Includes 10X Reaction Buffer and 10 mM ATP Solution. This endonuclease encoded by bacteriophage lambda recognizes and cleaves DNA at cos sites, generating 5´-overhangs of 12 bases in length. Since the 12-base cos site sequence is rare, the sizes of inserts in BAC, fosmid, or cosmid clones can be rapidly determined. The clones are linearized with lambda terminase and separated by pulsed field gel electrophoresis. Lambda Terminase can also be used for chromosomal mapping and for generating restriction maps of DNA cloned into BAC, cosmid, or fosmid vectors. •Specific cleavage of chromosomal DNA for physical mapping. Applications •Rapid sizing of BAC, fosmid, or cosmid clones. •Generation of restriction maps of BAC, cosmid, or fosmid clone inserts. •Linearization of cos site-containing clones for in vitro packaging. FIG 1. Digestion of BAC clones with Lambda Terminase gives one band while digestion with Not I often gives multiple bands. DNA from six randomly chosen BAC clones were digested with Not I (Panel 1) or Lambda Terminase (Panel 2) Lane M: Lambda Ladder PFG marker, Lane 1-6: BAC clones, Lane BT: BAC-Tracker™ Supercoiled Ladder. Properties of DNA Exonucleases DNA Exonucleases Enzyme Exonuclease I Substrate Activity Products Applications dNMPs Removal of ssDNA and oligonucleotides. 80°C for 15 min Used with S1 Nuclease or Mung Bean Nuclease to make nested deletions. Preparation of ssDNA templates for sequencing. Site-directed mutagenesis. Preparation of labeled strand-specific probes. 70°C for 20 min Removal of primers and single-stranded oligos. 95ºC for 10 min ssDNA 3´→ 5´ exonuclease Exonuclease III (E. coli) dsDNA 3´→ 5´ exonuclease that digests duplex DNA from the 3´-end of a nick or a blunt or 3´-recessed end; not active on thionucleotides. Exo III also has RNase H, 3´-DNA phosphatase and apurinic DNA endonuclease activities. Exonuclease VII ssDNA Exonuclease that digests in both 5´→ 3´ and 3´→ 5´ directions. Lambda Exonuclease dsDNA 5´→ 3´ exonuclease that digests dsDNA from 5´phosphorylated blunt or recessed ends. It has low activity on 5´-hydroxylated ends and is not active on nicked DNA. RecBCD Nuclease (E. coli) dsDNA and ssDNA An ATP- and Mg2+- dependent exonuclease that digests linear DNA in both 5´→ 3´ and 3´→ 5´ directions. Not active on nicked or closed-circular dsDNA. dNMPs Removal of linear DNA from circular DNA. Rec J Exonuclease ssDNA 5´→ 3´ exonuclease that digests ssDNA with a 5´phosphate or a 5´-OH. dNMPs Removal of primers and ssDNA from dsDNA. T5 Exonuclease dsDNA and ssDNA 5´→ 3´ exonuclease that also has single-strandspecific endonuclease activity in presence of 1-10 mM Mg2+ ions. At <1 mM Mg2+, the 5´→ 3´ exonuclease can digest from a nick in closed-circular dsDNA without digesting the opposite strand. dNMPs and ssDNA on the opposite strand. Partial digestion produces dsDNA having 5´ extensions of ssDNA. dNMPs dNMPs and ssDNA on the the opposite strand. Partial Preparation of ssDNA digestion produces templates for sequencing. dsDNA having 3´ extensions of ssDNA. dNMPs. Circular ssDNA is obtained from closed-circular dsDNA in presence of <1 mM Mg2+. Heat Inactivate Plasmid mutagenesis. Oligonucleotide site-directed mutagenesis. Removing linear DNA from plasmid preps. Preparation of circular ssDNA. 75°C for 10 min N/A 65°C for 20 min N/A Exonuclease I, E. coli Catalog No. Conc. Exonuclease I, E. coli X40501K 20 U/µl X40505K 20 U/µl 12 Size 1,000 U 5,000 U Exonuclease I digests single-stranded DNA (ssDNA) in a 3´→ 5´ direction, but does not digest double-stranded DNA (dsDNA). Although it requires the presence of magnesium and a free 3´-hydroxyl terminus for activity, it is active under a wide variety of buffer conditions and can be added directly into most reaction mixes. It can be heat-inactivated by incubation at 80°C for 15 minutes. Applications •Removal of residual ssDNA, including oligos, from reaction mixes. •Removal of ssDNA from nucleic acid mixtures. [email protected] • www.EpiBio.com Exonuclease III, E. coli •Production of intermediates for site-directed mutagenesis protocols. •Production of strand-specific radiolabeled probes. Catalog No. Conc. Exonuclease III, E. coli EX4405K 200 U/µl EX4425K 200 U/µl Size 5,000 U 25,000 U Nucleases Applications Exonuclease III digests duplex DNA in a 3´→ 5´ direction from a nick or a blunt or 3´-recessed end, producing stretches of single-stranded DNA on the opposite strand. Exonuclease VII Exonuclease VII has high enzymatic specificity for single-stranded DNA and exhibits both 5´→ 3´ and 3´→ 5´ exonuclease activities. It is especially useful for rapid removal of single-stranded oligonucleotide primers from a completed PCR reaction when different primers are required for subsequent PCR reactions. Exonuclease VII digestion of single-stranded DNA occurs in the absence of magnesium. Applications •Removal of single-stranded oligonucleotide primers after PCR. Catalog No. Conc. Exonuclease VII EN510100 10 U/µl EN510250 10 U/µl Size 100 U 250 U •Minimize the effect of primers left over from previous PCR reactions. Lambda Exonuclease Applications This highly processive 5´→ 3´ exodeoxyribonuclease selectively digests the phosphorylated strand of double-stranded DNA. The preferred substrate is blunt-ended, 5´-phosphorylated double-stranded-DNA. The enzyme has reduced activity against nicked DNA and against single-stranded DNA and gapped DNA. •SSCP (single-strand conformation polymorphism) analysis of PCR product. •Generate single-stranded DNA sequencing template from PCR product. Catalog No. Conc. Lambda Exonuclease LE035H 500 U LE032K 2,500 U Size 10 U/µl 10 U/µl Lambda Exonuclease (5'-phosphate-specific 5' 5'OH 3' exodeoxyribonuclease) OH P OH OH P OH P PCR using one primer with a 5'-phosphate 5'OH OH OH P 5'OH OH OH P PCR product with strand-specific 5'-phosphate Treatment with Lambda Exonuclease 5'OH 5'OH OH OH Single-stranded PCR product dNMPs FIG 1. Lambda Exonuclease selectively digests the strand of a PCR product produced using a PCR primer with a 5´-phosphate. The resulting single-stranded PCR product can be used for SSCP analysis or sequencing. RecBCD Nuclease, E. coli This exonuclease from E. coli degrades singleand double-stranded DNA. Hydrolysis of the DNA is bi-directional from both the 3´ and 5´ ends and processive, producing nucleoside monophosphates. Magnesium is required for the exonuclease activity, while calcium, nickel, zinc, and copper inhibit exonuclease activity. Calcium allows double-stranded DNA unwinding (helicase activity) without hydrolysis. www.EpiBio.com • [email protected] Applications •Removal of contaminating bacterial chromosomal DNA in plasmid, fosmid, cosmid, and BAC clone or vector preparation. Catalog No. Conc. RecBCD Nuclease, E. coli BCD0401K - Size 1,000 U 13 Nucleases Rec J Exonuclease Catalog No. Conc. Rec J Exonuclease RJ411050 10 U/µl RJ411250 10 U/µl Size 50 U 250 U Rec J Exonuclease, derived from E. coli, catalyzes removal of deoxyribonucleoside monophosphates from single-stranded DNA in a 5´→ 3´ direction. Its activity is dependent on Mg+2. Rec J Exonuclease can be heatinactivated by incubation at 65°C for 20 minutes. Applications This highly efficient 5´→ 3´ exonuclease for either single-stranded or duplex DNA has a tightly associated single-strand-specific endonuclease activity when used in the presence of 1-10 mM divalent magnesium ions. This activity may be selectively suppressed by using low concentrations of magnesium ions (< 1 mM), allowing nicked, double-stranded circular DNA to be “gapped” to a singlestranded circular species. In the absence of divalent metal cofactors, T5 Exonuclease is able to bind to DNA with a single-strand arm adjacent to a duplex DNA region. •Removes primers from completed PCR reactions. •Degrades single-stranded linear DNA in dsDNA and plasmid preps. T5 Exonuclease Catalog No. Conc. T5 Exonuclease T5E4111K 10 U/µl Size 1,000 U •Plasmid mutagenesis methods. •Oligonucleotide site-directed mutagenesis. •Generation of plasmid-sequencing templates. •Removal of denatured DNA from alkalinebased plasmid purification procedures for improved cloning procedures. Our Exclusive RNA Exonuclease RNase R Catalog No. RNase R RNR07250 Applications Conc. - Size 250 U Ribonuclease R is a magnesium-dependent 3´→ 5´ exoribonuclease that digests essentially all linear RNAs but will not digest lariat or circular RNA structures. Intron RNA can be isolated from total RNA samples by digestion with RNase R. After digestion, only lariat structures that are produced during pre-mRNA splicing of intron regions remain. Applications •Alternative splicing studies. •Gene expression studies. •Intron cDNA production. •Intronic screening of cDNA libraries. Ribonuclease R (RNase R) Pre-mRNA Exon 1 Intron Exon 2 Intron Exon 1 Exon 2 Intron mRNA Exon 1 Exon 2 Intron Lariat Only Remains i2220706 RNase R Digestion FIG 1. How RNase R works. 14 [email protected] • www.EpiBio.com Properties of RNA Endonucleases Substrate RNase A ssRNA Cleaves ssRNA 3´ of pyrimidine residues. ssRNA Cleaves ssRNA between all dinucleotide pairs. RNase III, E. coli dsRNA Cleaves dsRNA in presence of Mg2+ to 12-15-bp dsRNA. Cleaves dsRNA in presence of 20 mM Co2+ or Mn2+ to 18-25-bp dsRNA. dsRNA with 5´phosphate and 2-base 3´-overhangs with 3´-OH Random cleavage of long dsRNA to short dsRNA. RNA interference (RNAi). RNase H, E. coli RNA in RNA:DNA hybrid Cleaves RNA in RNA:DNA hybrid without affecting unhybridized RNA or DNA. oligoribonucleotides with 5´-phosphate and 3´-OH Elimination of RNA prior to second strand cDNA synthesis. Removal of poly(A) tails from mRNA hybridized to oligo(dT). Hybridase™ Thermostable RNase H RNA in RNA:DNA hybrid Cleaves RNA in RNA:DNA hybrid without affecting unhybridized RNA or DNA. oligoribonucleotides with 5´-phosphate and 3´-OH High-stringency hybrid selection. Highstringency mapping of mRNA structure. Transcription-based amplification methods. No RNA mapping and structure studies. Removal of RNA from DNA preps. N/A Removal of all RNA from genomic and cloned DNA preps. No RNase I, E. coli Activity Products Heat Inactivate Enzyme Applications oligoribonucleotides with 3´-cytidine or 3´-uridine residues Removal of RNA from DNA preps. RNase protection assays. RNA mapping and structure studies. N/A NMPs with 5´-OH and 2’,3´-cyclic monophosphate Removal of RNA from DNA preps. RNase protection assays. Mismatch detection of single basepairs in RNA:RNA or RNA:DNA hybrids. 70°C for 15 min RNase T1, Aspergillis oryzae ssRNA Cleaves ssRNA 3´ of GMPs. oligoribonucleotides with 3´-GMP residues RiboShredder™ RNase Blend ssRNA Efficiently degrades all RNA. NMPs Nucleases RNA Endonucleases No 5´phosphate Studies on RNA structure, RNA processing and maturation. 65°C for 10 min RNase A RNase A is an endoribonuclease that cleaves single-stranded RNA at the 3´-end of pyrimidine residues, forming oligoribonucleotides having 3´terminal pyrimidine-3´-phosphates. Pyrimidine3´-monophosphates are also released by RNase A cleavage of adjacent pyrimidine nucleotides. Modified RNA containing pyrimidine-2’-fluorodNMPs, such as modified RNA made by in vitro transcription using EPICENTRE’s DuraScribe® T7 & SP6 Transcription Kits is completely resistant to cleavage by RNase A. Catalog No. Conc. Ribonuclease A MRNA092 - Applications Size 2 ml @ 5 mg/ml •Removal of RNA from DNA preparations. •Removal of unhybridized regions of RNA from DNA-RNA or RNA-RNA hybrids. RNase I, E. coli RNase I degrades single-stranded RNA to nucleoside 3´-monophosphates via 2’, 3´ cyclic monophosphate intermediates by cleaving between all dinucleotide pairs. This enzyme is completely inactivated by heating at 70°C for 15 minutes, eliminating the requirement to remove the enzyme prior to many subsequent procedures. Applications •Removal of RNA from DNA preparations. •RNase protection assays to detect singlebasepair mismatches in RNA:RNA and RNA:DNA hybrids. Catalog No. Conc. RNase I, E. coli N6901K 10 U/µl N6905K 10 U/µl Size 1,000 U 5,000 U Provided with Dilution Buffer. RNase III, E. coli This endoribonuclease specifically digests double-stranded RNA (dsRNA) to dsRNA fragments that have 2-base, 3´-overhangs. Complete digestion of dsRNA results in dsRNA fragments of 12-15 bp. www.EpiBio.com • [email protected] Applications •Digest long dsRNA to short dsRNA. •RNA structure studies. Catalog No. Conc. RNase III, E. coli RN02950 1 U/µl Size 50 U • RNA processing and maturation studies. 15 Nucleases RNase H, E. coli Catalog No. Conc. RNase H, E. coli R52250 10 U/µl R0601K 10 U/µl Size 250 U 1,000 U This endonuclease specifically degrades the RNA in an RNA:DNA hybrid, without affecting DNA or unhybridized RNA. It will not degrade double-stranded DNA or single-stranded DNA or RNA. E. coli RNase H is inactivated at 55°C in 20 minutes. Applications •Elimination of RNA prior to second-strand synthesis of cDNA. •Removal of poly(A) tails from messenger RNA hybridized to oligo(dT). •Specific cleavage of mRNAs after hybridization to oligonucleotide probes. •Specific destruction of “hybrid-arrested” mRNAs during in vitro translation. •Diagnostic assays using NASBA™ transcription-based amplification methods. •Diagnostic assays based on the Cycling Probe Technology. •Template–dependent probe technologies. Hybridase™ Thermostable RNase H* Catalog No. Conc. Size Hybridase™ Thermostable RNase H H39100 100 U H39500 500 U EPICENTRE’s patented Hybridase™ Thermostable RNase H specifically degrades the RNA in a DNA:RNA hybrid, without affecting DNA or unhybridized RNA. It has optimal activity above 65°C and can be used at temperatures up to 95°C. The thermostability of the enzyme permits it to be used at temperatures that give the highest hybridization stringency for specific DNA:RNA heteroduplexes, maximizing sensitivity and selectivity while minimizing background due to nonspecific hybridization. Applications This endoribonuclease specifically cuts RNA or deaminated RNA at the 3´-end of guanosine residues and adjacent nucleotides through a 2’, 3´-cyclic phosphate intermediate mechanism. Applications •High stringency hybrid selection. •Diagnostic assay of specific target DNA sequences by isothermal probe amplification. •Transcription-based amplification methods (e.g., NASBA™). •High stringency mapping of mRNA structure. •Applications that require specific hydrolysis of the RNA in a DNA:RNA hybrid. *Hybridase™ Thermostable RNase H is covered by U.S. Patent Nos. 5,268,289; 5,459,055; and 5,500,370 assigned to EPICENTRE. This product is accompanied by a limited non-exclusive license for the purchaser to use the purchased product solely for life science research. Contact EPICENTRE for information on licenses to other uses. RNase T1, Aspergillus oryzae Catalog No. Conc. Size RNase T1, Aspergillus oryzae NT09100K 100,000 U NT09500K 500,000 U •RNA mapping and structure studies. •Removal of RNA from DNA preparations. •RNA protection assays. RiboShredder™ RNase Blend Catalog No. Conc. Size RiboShredder™ RNase Blend RS12100 100 U RS12500 500 U RiboShredder™ is a cocktail of potent RNases that completely degrades unwanted RNA in DNA purification procedures. This highly active cocktail contains a proprietary optimized blend of non-mammalian RNase enzymes. RiboShredder RNase Blend degrades all RNA, converting RNA to nucleoside monophosphates. • Completely degrades RNA rapidly. • DNase-free. Applications •Removal of RNA from genomic and cloned DNA preparations. 16 FIG 1. Comparison of RNA-degrading capability of RiboShredder™ RNase Blend versus commonlyused individual RNases or other RNase cocktails. Nucleic acids from a standard alkaline lysis plasmid preparation were treated as follows under standard reaction conditions: Lane 1, RNase A; Lane 2, RNase I; Lane 3, RNase T1, Lane 4, RiboShredder RNase Blend; Lane 5, RNase A/RNase T1 cocktail; Lane 6, Untreated Alkaline lysis plasmid prep; Lane 7, supercoiled DNA ladder. [email protected] • www.EpiBio.com Enzyme 8-Oxoguanine-DNA Excision Mix (8-Oxo-G-DNA glycosylase or Fpg and Endonuclease IV) Uracil-DNA Excision Mix (HK™-UNG and Endonuclease IV) HK™-UNG Thermolabile UracilN-Glycosylase (UNG) (UNG is also called Uracil-DNA Glycosylase or UDG) Activity Products Applications Heat Inactivate Cleaves sugar-phosphate bond 5´ of 8-oxo-GMPs. dsDNA with single-nucleotide gaps where 8-oxo-G residues have been removed, or ssDNA strands with lengths equal to distances between 8-oxo-G residues. Site-specific cleavage of DNA at 8-oxo-G sites. Base Excision Sequence Scanning of DNA containing 8-oxo-GMPs. N/A Cleaves sugar-phosphate bond 5´ of dUMPs. dsDNA with single-nucleotide gaps where dUMP residues have been removed, or ssDNA strands with lengths equal to distances between dUMP residues. Fragmentation of dUMPcontaining DNA. Site-specific cleavage of DNA at dUMP sites. Base Excision Sequence Scanning of DNA containing dUMP residues. 65°C for 10 min Uracil base and abasic DNA. Abasic sites can subsequently be cleaved by AP lyases, such as Endonuclease IV, or by treatment with heat or alkaline bases. Fragmentation of dUMPcontaining DNA. Site-specific cleavage of DNA at dUMP sites. Base Excision Sequence Scanning of DNA containing dUMP residues. 65°C for 10 min Substrate dsDNA or ssDNA containing 8-oxo-GMP dsDNA or ssDNA containing dUMP dsDNA or ssDNA containing dUMP Removes uracil base from dUMP residues in DNA. Nucleases Properties of DNA Glycosylases and Excision Mixes Base-Specific DNA Excision Mixes and DNA Glycosylases 8-Oxoguanine-DNA Excision Mix 8-Oxoguanine-DNA Excision Mix is a blend of enzymes that allows site-specific or random cleavage of DNA at oxidized guanine residues. The positions of the oxidized guanine residues in a DNA sequence can be mapped by sizing cleavage fragments on a sequencing-type gel from a fixed priming site, yielding data similar to a G-lane dideoxy-sequencing reaction. The enzyme mix, first depurinates oxidized G-residues, then cleaves the deoxyribose phosphate backbone at apurinic sites, generating a “polished” 3´-hydroxyl end and releasing a DNA fragment with an abasic 5´phosphorylated end. The minimum oligomer size that will serve as a substrate for excision by the 8-Oxoguanine-DNA Excision Mix is 6 base pairs. The resulting DNA fragments can be analyzed by denaturing agarose gel or polyacrylamide gel electrophoresis. Catalog No. Conc. Size 8-Oxoguanine-DNA Excision Mix OG51100 - 100 Reactions Contents: 8-Oxoguanine-DNA Excision Enzyme Mix, Guanine Oxidation Reagent, and 10X 8-OxoG-DNA Excision Reaction Buffer. Applications •Mapping of G residues in any DNA. •DNA repair studies. Uracil-DNA Excision Mix Uracil-DNA Excision Mix is a blend of enzymes that cleave DNA at positions where uracil is present in place of thymine. The Uracil-DNA Excision Mix is useful for specific or random cleavage of DNA or for DNA repair studies, allowing mapping of uracil residues in any DNA. Uracil-DNA glycosylase in the Excision Mix removes uracil bases from DNA, creating a single base gap and leaving the deoxyribose phosphate backbone intact. Endonuclease IV in the Excision Mix then cleaves the DNA at each abasic site, leaving a 3´-hydroxyl end and an abasic 5´-phosphorylated end. The minimum oligomer size that will serve as a substrate is 6 base pairs. Uracil-DNA Excision Mix digestion products can be analyzed by denaturing agarose gel electrophoresis or denaturing polyacrylamide gel electrophoresis. Catalog No. Conc. Size Uracil-DNA Excision Mix UEM04100 - 100 Reactions Contents: Uracil-DNA Excision Enzyme Mix and 10X Uracil Excision Enzyme Buffer. Applications •Mapping of uracil-containing residues in any DNA. • Mapping CpG islands. • DNA repair studies. HK™-UNG Thermolabile Uracil N-Glycosylase This Uracil N-Glycosylase (also known as uracilDNA glycosylase) hydrolyzes the N-glycosidic bond between the deoxyribose sugar and uracil in DNA containing deoxyuridine in place of thymidine. HK-UNG is active on both single- and double-stranded DNA that contains uracil, but has no activity on RNA or 2’-deoxyuridine-5´monophosphate. www.EpiBio.com • [email protected] Fully active at 50°C; inactivated by a 10-minute incubation at 65°C. Applications •Repair studies of abasic sites in doublestranded DNA. Catalog No. Conc. Size HK™-UNG Thermolabile Uracil N-Glycosylase HU59100 1 U/µl 100 U HU5901K 1 U/µl 1,000 U Provided with Dilution Buffer. 17 Ligases Properties of Ligases Base-Specific DNA Excision Mixes and DNA Glycosylases Name of Ligase Ligation Temp Cofactor Type of Ends Ligated Fast-Link™ DNA Ligase Ligation Template Required to Ligate Blunt Cohesive Heat Inactivate Primary Application 5-15’ @ 20°C ATP NO* YES YES Rapid Cloning 10’ @ 65°C T4 DNA Ligase 4°C - 25°C ATP NO* YES YES Cloning 15’ @ 65°C E. coli DNA Ligase 5°C - 20°C NAD YES; DNA Only Weak Activity YES Make long cDNA 20’ @ 65°C Ampligase DNA Ligase 20°C - 95°C NAD YES; DNA Only NO YES TemplateDependent Ligation NO; Half-life 1 hr @ 95°C CircLigase™ ssDNA Ligase 20°C - 65°C ATP NO Ligates ssDNA N/A Make ssDNA Circles 5´ @ 100°C Ligate RNA to DNA Make chimeric DNA and RNA 15’ @ 65°C ® T4 RNA Ligase 37°C ATP NO Ligates ssDNA *These enzymes ligate blunt ends of dsDNA, but ligation is more efficient on a ligation template, which can be DNA or RNA. Fast-Link™ DNA Ligation Kit Catalog No. Conc. Size Fast-Link™ DNA Ligation Kit LK11025 25 Ligations LK0750H 50 Ligations Contents: Fast-Link™ DNA Ligase, Fast-Link™ 10X Ligation Buffer, 10 mM ATP *For a full line of cloning products, please visit www.EpiBio.com/cloning.asp. This kit uses a high-quality ligase, called Fast-Link™ DNA Ligase, that was cloned at EPICENTRE and then formulated to provide extremely rapid high-efficiency DNA ligation. Cohesive-end ligations can be performed in 5 minutes at room temperature. It can be used for routine and high-throughput DNA cloning. • Cohesive-end ligations in 5 minutes at room temperature. •Blunt-end ligations in 15 minutes at room temperature •Ligation of PCR product with A-overhangs in 1 hour at 16°C. •Desalting of ligation products is not needed prior to transformation. •High efficiency in-gel ligation. T4 DNA Ligase is a commonly used ATPdependent ligase for DNA cloning. It covalently joins double-stranded DNA molecules having 5´-phosphorylated and 3´-hydroxylated blunt or compatible cohesive ends produced by restriction enzyme digestion or other enzymatic processes. T4 DNA Ligase has no activity on single-stranded nucleic acids. Following a ligation reaction, T4 DNA Ligase may be inactivated by incubation at 65°C for 10 minutes. EPICENTRE’s T4 DNA Ligase is the highest quality T4 DNA Ligase commercially available. This NAD+-dependent enzyme catalyzes the formation of phosphodiester bonds between complementary 3´-hydroxyl and 5´-phosphoryl termini of double-stranded DNA. The enzyme works best with cohesive dsDNA ends and is also active on nicked DNA. Blunt ends can be ligated in the presence of condensing reagents such as polyethylene glycol or Ficoll. It is not effective for formation of DNA-RNA or RNA-RNA hybrids. Applications T4 RNA Ligase catalyzes the formation of a phosphodiester bond between a 5´-phosphorylterminated nucleic acid donor and a 3´-hydroxyl nucleic acid. The enzyme is active RNA, DNA, oligoribo and oligodeoxyribonucleotides, and nucleotide derivatives. Applications Applications •TA cloning. •PCR blunt-end cloning. •Genomic DNA cloning and subcloning. •BAC library construction. •cDNA cloning. •Linker ligation. T4 DNA Ligase, Cloned Catalog No. Conc. T4 DNA Ligase, Cloned L0805H 2 U/µl L0810H 2 U/µl LH805H 10 U/µl LH810H 10 U/µl Size 500 U 1,000 U 500 U 1,000 U Includes 10X Reaction Buffer and a separate 25 mM ATP Solution. *For a full line of cloning products, please visit www.EpiBio. com/cloning.asp. Applications •Ligation of blunt or cohesive-ended DNA fragments. •Repair of nicks in double-stranded nucleic acids. E. coli DNA Ligase Catalog No. Conc. E. coli DNA Ligase DL04082H 10U/µl Size 200 U •Molecular cloning of dsDNA with cohesive ends. •Blunt-end ligation in presence of 10-15% PEG and high concentrations of monovalent cations. •cDNA cloning of products from second strand cDNA synthesis experiments. T4 RNA Ligase Catalog No. T4 RNA Ligase LR5010 LR5025 Conc. 5 U/µl 5 U/µl Size 1,000 U 2,500 U Includes 10X Reaction Buffer and a 10 mM ATP Solution. •mRNA tagging to map and sequence 5´termini or as a step in cDNA synthesis (RACE). •3´-End labeling of RNA species. •Ligation of RNA and DNA species to form circles, extended oligonucleotides, or RNADNA-containing oligonucleotides. •Modifications or mutagenesis of RNA species. 18 [email protected] • www.EpiBio.com Ampligase® Thermostable DNA Ligase oligonucleotide multimers when nucleotide repeats are present in a DNA template. •Simultaneous mutagenesis of multiple sites: Ampligase DNA Ligase can introduce single or multiple point mutations at specific sites by ordered ligation of PCR-amplified DNA fragments that have had point mutations introduced via mutant primers. •Other ligation-based detection methods. Ampligase® Enzyme and Buffer A0102K 100 U/µl 2,500 U A32250 5 U/µl 250 U A3202K 5 U/µl 2,500 U Ampligase® DNA Ligase A0110K 100 U/µl A0125K 100 U/µl A3210K 5 U/µl A3225K 5 U/µl High specificity and stringency permits sensitive detection of SNPs. 10,000 U 25,000 U 10,000 U 25,000 U One unit of Ampligase™ is equal to as many as 15 units of other thermostable DNA ligases. Please compare competitive unit definitions. Supplied as enzyme only; Reaction Buffer is not included. Applications •Repeat Expansion Detection (RED): RED is a ligation-based method of genetic screening that detects DNA regions comprised of multiple nucleotide repeats. Ampligase DNA Ligase is used in a two-step thermal cycling reaction that generates One unit of Ampligase™ is equal to as many as 15 units of other thermostable DNA ligases. Please compare competitive unit definitions. Contains Ampligase® DNA Ligase, Ampligase® 10X Reaction Buffer, and Ligation Control DNA. One unit of Ampligase™ is equal to as many as 15 units of other thermostable DNA ligases. Please compare competitive unit definitions. 25 µl of Ampligase® 10X Reaction Buffer is supplied with each 50 units of Ampligase® DNA Ligase. High thermostability allows ligation using highstringency hybridization conditions. •Ligation Amplification (Ligase Chain Reaction, LCR): Ligation Amplification can distinguish between DNA sequences that differ by as little as a single base-pair and is a useful tool for detection of single nucleotide polymorphisms (SNPs). Catalog No. Conc. Size Ampligase® DNA Ligase Kit A8101 5 U/µl 1,000 U A30201 5 U/µl 5,000 U Ligases Derived from a thermophilic bacterium, stable and active at much higher temperatures than conventional DNA ligases, this enzyme catalyzes NAD-dependent ligation of adjacent 3´-hydroxylated and 5´-phosphorylated termini in duplex DNA structures that are stable at high temperatures. Its half-life is 48 hours at 65°C and greater than 1 hour at 95°C. It has been shown to be active for at least 500 thermal cycles (94°C/80°C) or 16 hours of cycling, which permits extremely high hybridization stringency and ligation specificity. No detectable activity in ligating blunt-ended DNA, RNA or RNA:DNA hybrids. X X X Ampligase® 10X Reaction Buffer A1905B 5 ml Ampligase® 1X Storage Buffer A3201S - 1 ml X FIG 1. Schematic of mutation discovery and screening using ligation amplification. The existence of a point mutation at the site of ligation interferes with oligonucleotide ligation, resulting in no ligation product. The lack of an amplification product indicates the presence of a point mutation at the ligation site. Oligos can also be designed so ligation occurs in the presence of the mutant template. CircLigase™ ssDNA Ligase This thermostable ATP-dependent ligase catalyzes intramolecular ligation (i.e., circularization) of single-stranded DNA (ssDNA) templates having a 5´-phosphate and a 3´hydroxyl group and ligates ends of ssDNA in the absence of a complementary sequence. It is therefore useful for making circular ssDNA molecules from linear ssDNA. Circular ssDNA molecules can be used as substrates for rolling circle replication or rolling circle transcription. Efficient single-stranded DNA ligase activity. Circularizes single-stranded DNA of >30 bases. Standard reaction conditions produce no detectable single-stranded DNA concatamers or concatameric DNA circles. Applications •Production of single-stranded DNA templates for rolling circle replication or rolling circle transcription experiments. www.EpiBio.com • [email protected] •Production of single-stranded DNA templates for RNA polymerase and RNA polymerase inhibitor assays. FIG 1. CircLigase™ ssDNA Ligase converts linear ssDNA to circular ssDNA. A 71-base ssDNA oligo was converted to a circular DNA form in a reaction containing CircLigase™ ssDNA Ligase and ATP. Lane M, DNA markers. Lane 1, 71-base ssDNA. Lane 2, circularization proceeds through an adenylated intermediate. Lane 3, the closed circular nature of the reaction product was confirmed by treating the reaction with exonuclease I, which specifically digests linear DNA. Catalog No. Conc. Size CircLigase™ ssDNA Ligase CL4111K 1,000 U CL4115K 5,000 U Contents: CircLigase™ ssDNA Ligase, CircLigase™ 10X Reaction Buffer, ATP, 50 mM MnCl2 CircLigase™ Linear ssDNA Control Substratem, Water *Circligase™ ssDNA Ligase is covered by intellectual property rights licensed to EPICENTRE. The purchase of this product conveys to the buyer the non-transferable right to use the purchased product and components of the product in research conducted by the buyer. The buyer cannot sell or otherwise transfer this product or its components to a third party and in particular, no rights are conveyed to the buyer to use the product or its components for commercial use purpose other than for research to gain information that is used by the buyer. 19 Phosphatases and Kinase Properties of EPICENTRE’S Phosphatases Heat-Labile Alkaline Phosphatases Phosphatase APex™ Type of Ends Dephosphorylated Reaction Temp Nucleotides are Substrates Active at pH Heat Inactivate YES YES 5.5-12 5´ @ 70°C - YES ~7-10 15’ @ 65°C Reaction Time 5´Protruding Blunt 5´Recessed 20° - 50°C Optimal @ 37°C YES YES 37°C Varies with Substrate Amt - - NTPhos™ APex™ Heat-Labile Alkaline Phosphatase Catalog No. Conc. Size APex™ Heat-Labile Alkaline Phosphatase AP49010 1 Reaction/µl 10 Reactions AP49050 1 Reaction/µl 50 Reactions This is a new, innovative enzyme preparation with improved performance over other alkaline phosphatases. APex™ Phosphatase removes the 5´-phosphate from all types of DNA ends, including 5´ protruding, blunt, and 5´ recessed ends, and from RNA ends. The enzyme is irreversibly heat-inactivated by incubation at 70°C for 5 minutes. active over a wide range of temperatures, pH, salts and buffers. •Active on blunt, 5´- and 3´-overhang restricted DNA ends for compatibility with any restriction enzyme or experimental design. •One simple protocol for most applications. Applications •Fast, complete and irreversible heatinactivation for easy transition to next step; no time-consuming substrate purification with phenol:chloroform extraction. •Dephosphorylation of DNA vectors prior to cloning to prevent recircularization. •Flexible and easy to use—add directly to most RE buffers without supplementation; •Dephosphorylation of DNA/RNA substrates for other purposes. The 5´-termini of many natural RNA molecules, including most eukaryotic messenger RNAs, viral RNAs, many small nuclear RNAs, and heterogeneous nuclear RNAs, have a structure called a “cap.” Tobacco Acid Pyrophosphatase (TAP) hydrolyzes the phosphoric acid anhydride bonds in the triphosphate bridge of the cap structure, releasing the cap nucleoside and generating a 5´-phosphorylated terminus on the RNA molecule. The resulting “decapped” 5´-phosphorylated terminus may be ligated to a 3´-hydroxylated terminus using T4 RNA Ligase or dephosphorylated using APex™ Heat-Labile Alkaline Phosphatase for end labeling. Similarly, TAP digests the triphosphate group at the 5´-end of prokaryotic transcripts, generating an RNA molecule with a 5´-phosphorylated terminus. •5´ and 3´-end mapping of RNA. Applications FIG 1. How TAP works •Preparation of 5´-nucleic acid termini for 5´end labeling with polynucleotide kinase. Tobacco Acid Pyrophosphatase •Ligation of oligoribonucleotides to TAP-treated cellular RNA for construction of full-length cDNA libraries. •Mapping of transcription initiation sites for eukaryotic and prokaryotic transcripts. •Radiolabeling of RNA for use in sequencing or as a hybridization probe. GpppG OH Capped RNA TAP Treatment OH Decapped RNA pG RNA Ligase •Preparation of templates for RACE (Rapid Amplification of cDNA Ends). pG Ligated RNA + T4 Polynucleotide Kinase, Cloned Catalog No. Conc. Size T4 Polynucleotide Kinase, Cloned P0505H 10 U/µl 500 U P0501K 10 U/µl 1,500 U Includes 10X Reaction Buffer without ATP. ATP is available separately. ATP Solution R109AT - 5 µmoles Provided as 500 µl of a 10 mM solution, pH 7.0. 20 pG T4 Polynucleotide Kinase (PNK) catalyzes the transfer of the gamma-phosphate from ATP to the 5´-hydroxyl of single- and double-stranded DNA, RNA, and nucleoside 3´-monophosphates. The enzyme also removes the 3´-phosphate from 3´-phosphoryl polynucleotides, deoxyribonucleoside 3´-monophosphates, and deoxyribonucleoside 3´, 5´-diphosphates to form a 3´-hydroxyl group. Applications pG OH + additional ligation products • L abeling of 5´-termini of DNA and RNA for DNA sequencing, blot-hybridization, or transcript mapping. •Phosphorylation of oligonucleotide linkers and other DNA or RNA molecules prior to ligation, or for use in ligation amplification with Ampligase® Thermostable DNA Ligase. •Preparation of labeled DNA or RNA molecular weight markers for gel electrophoresis and chromatography. [email protected] • www.EpiBio.com i490407tap2 Catalog No. Conc. Size Tobacco Acid Pyrophosphatase (TAP) T19050 50 U T19250 250 U EasyLyse™ Bacterial Protein Extraction Solution • Rapid protein screening. •Easy protein purification. • Enzymatic studies. • ELISA studies. •Manual or robotic procedures. Bacterial Lysis (%) 60 40 0 EasyLyse™ Supplier P Contents: Lysis Buffer, Enzyme Mix, MgCl2 Solution. c170308bnl 20 Catalog No. Conc. Size EasyLyse™ Bacterial Protein Extraction Solution 500, 1-ml Purifications or RP03750 48, 96-well Microplates, 100 µl/well FIG 2. E. coli Lactate Dehydrogenase (LDH) specific activity expressed as nmol/min/µg of soluble cell protein. EasyLyse™ Preserves Enzyme Activity 1.5 1.2 1.0 0.5 0.12 0.0 EasyLyse™ Supplier P c180308bnl Applications 80 Specific Activity of Soluble LDH (nmol/min/µg) Gives higher yields of soluble protein. FIG 1. Lysis of E. coli aliquots with soluble protein quantities expressed as a % of total protein, as determined by the Coomassie Plus Protein Assay. EasyLyse™ Bacterial Lysis Efficiency 100 Lysozymes This extraction solution is designed for lysing bacterial cells for the isolation of proteins, especially recombinant gene products expressed in E. coli, without significant loss of enzymatic activity. It contains a highly active enzyme for cell lysis and a potent nuclease that reduces extract viscosity by digesting all nucleic acids in the sample. The EasyLyse Solution is formulated as a homogeneous reagent for ease of use in high-throughput applications without sonication. Table 1. Bacteria lysed with Ready-Lyse Lysozyme Solution. Gram-negative Gram-positive Escherichia coli Salmonella typhimurium Actinobacillus pleuropneumoniae Rhodobacter sphaeroides Shewanella putrefaciens Flavobacteria odoratum Oerskovia xanthinolytica Bacillus subtilis Catalog No. Conc. Size Ready-Lyse™ Lysozyme Solution R1802M 2 X 106 U R1810M 10 X 106 U *Animal Product-Free Ready-Lyse™ Lysozyme Solution is available. **For a complete line of purification products, please visit www.EpiBio.com. Applications •Lysis of Gram-negative or Gram-positive bacteria for protein purification. 3 hr. M 1 hr. Ready-Lyse™ Lysozyme Solution is a nonmammalian, non-avian, recombinant lysozyme preparation for the lysis of Gram-negative (such as E. coli) and Gram-positive (such as Bacillus sp.) bacteria. The specific activity of Ready-Lyse Lysozyme is 200-fold higher than the specific activity of egg white lysozyme. Also, unlike egg white lysozyme, Ready-Lyse Lysozyme Solution is stable at –20°C, eliminating the need to prepare a fresh solution for each use. The use of Ready-Lyse Lysozyme results in higher yields of protein than can be obtained with standard egg white lysozyme. 0 hr. Ready-Lyse™ Lysozyme Solution for Protein Extraction 1 2 3 4 FIG 1. Use of Ready-Lyse™ Lysozyme Solution to recover recombinant proteins. One ml of induced cells from a recombinant E. coli clone was pelleted by microcentrifugation before induction and at 1 and 3 hours after induction. Ready-Lyse™ Lysozyme Solution for Nucleic Acid Extraction Ready-Lyse™ Lysozyme Solution is a nonmammalian, non-avian, recombinant lysozyme preparation for the lysis of many Gram-negative bacteria such as Escherichia coli and Grampositive bacteria such as Bacillus subtilis. The use of Ready-Lyse Lysozyme results in higher yields of DNA and RNA than obtained with standard egg white lysozyme. that are used once and then discarded, as is required with egg white lysozyme. Due to its higher specific activity, less ReadyLyse is needed for lysis compared to egg white lysozyme. This reduces loss of nucleic acid from enzyme binding. FIG 1. Lysis with Ready-Lyse™ Lysozyme increases yields of nucleic acids. Approximately 50% of the DNA was lost due to precipitation by egg white lysozyme (EW), while Ready-Lyse Lysozyme (RL) caused minimal precipitation losses of DNA compared to control (C) samples without lysozyme. Supplied as a ready-to-use solution stable at –20°C, so there is no need to prepare fresh solution prior to each use or to freeze aliquots www.EpiBio.com • [email protected] Applications •Lysis of Gram-negative and Gram-positive bacteria for preparations of nucleic acids. •In-well gel screening of recombinant DNA in agarose gels. Catalog No. Conc. Size Ready-Lyse™ Lysozyme Solution R1802M 2 X 106 U R1810M 10 X 106 U *Animal Product-Free Ready-Lyse™ Lysozyme Solution is available. **For a complete line of purification products, please visit www.EpiBio.com. Red Cell Lysis Solution MRC0912H 1200 ml Tissue & Cell Lysis Solution MTC096H 600 ml 21 Other Protein products RecA Protein, E. coli Catalog No. Conc. RecA Protein, E. coli RC44200 5 µg/µl RC441MG 5 µg/µl Size 200 µg 1 mg This multi-functional DNA-binding protein encoded by E.coli plays integral roles in both homologous recombination and post-replicative DNA repair mechanisms. In vitro, RecA Protein helps promote homologous recombination through a multiple-step ATP-dependent pathway. Initially, the protein binds preferentially to single-stranded DNA forming a nucleoprotein filament. The filament complex binds to naked duplex DNA and searches for regions of homology. Once a region of homology is found, strand displacement and exchange begins. Applications •Site-directed mutagenesis through displacement loop structures. •Targeted site-specific cleavage of small and large DNA. •Enrichment of target sequences from libraries or other DNA pools. •Visualization of DNA for electron microscopy. •Cloning or other experiments involving use of RecA Protein and a site-specific oligonucleotide to block endonuclease cleavage at the complementary site in a target DNA molecule. Single-Stranded DNA Binding Protein (SSB), E. coli Catalog No. Conc. Size Single-Stranded DNA Binding Protein (SSB) SSB02200 2 mg/ml 200 µg Single-Stranded DNA Binding binds singlestranded DNA with high specificity. In vivo, SSB is involved in DNA replication, recombination, and repair. In vitro, SSB enhances several molecular biology applications by destabilizing DNA secondary structure and increasing the processivity of polymerases. E. coli SSB is also required for in vitro transcription of single-stranded DNA templates by MiniV™ RNA Polymerase, a transcriptionally-active 1,106-amino acid domain of the N4 virion RNA polymerase. Applications •Transcription of ssDNA templates by MiniV™ RNAP. •Targeting restriction endonuclease digestion to any restriction enzyme site in cloned single-stranded DNA. •Enhance the specificity and yield of PCR reactions. •Improve DNA sequencing results through regions with strong secondary structure. •Site-directed mutagenesis when used in conjunction with recA protein. •Improve the processivity of DNA polymerases. •DNA replication and recombination studies. DNA Topoisomerase I, Vaccinia Catalog No. Conc. Size DNA Topoisomerase I, Vaccinia VT710500 10 U/µl 500 U VT7105K 10 U/µl 5,000 U 22 Topoisomerase I from vaccinia virus is a type I eukaryotic topoisomerase that removes both positive and negative superhelical turns (also called right- and left-handed supercoils) from covalently closed DNA. The product of the reaction is a covalently closed, circular DNA with fewer positive or negative superhelical turns. DNA Topoisomerase I does not absolutely require Mg2+ to function, although low concentrations of magnesium ions may increase activity. Applications •Studying the effects of supercoiling on transcription in vitro. •Studying chromatin reconstitution in vitro. •Determining the degree of supercoiling of naturally occurring DNA. •Detecting mutant plasmids that differ in length by only one basepair. •Increasing restriction endonuclease digestion of resistant DNA substrates by “unwinding” the DNA coils to expose restriction sites. [email protected] • www.EpiBio.com Tagetin™ RNA Polymerase Inhibitor Applications • RNA polymerase studies. • Transcription studies. Catalog No. Conc. Size Tagetin™ RNA Polymerase Inhibitor T9705H 20 U/µl 500 U T9702K 20 U/µl 2,500 U FIG 1. Tagetin Inhibitor activity on E. coli RNA Polymerase. Each gel lane shows products of a standard transcription reaction using a bacteriophage template, 1 U of E. coli RNA Polymerase Holoenzyme, and varying amounts of Tagetin Inhibitor. Lane 1, 100 U; Lane 2, 10 U; Lane 3, 1 U; Lane 4, 0.1 U; Lane 5, control without Tagetin Inhibitor. 50% inhibition of transcription is seen at 1 U Tagetin Inhibitor per unit of E. coli RNA Polymerase. Enzyme and Protein Inhibitors Tagetin™ RNA Polymerase Inhibitor is the only compound known to potently and selectively inhibit RNA polymerase III from a variety of eukaryotic organisms including mammalian cells, Saccharomyces cerevisiae, Drosophila melanogaster, Bombyx mori, and Xenopus laevis oocytes. It strongly inhibits Escherichia coli RNA polymerase and plant chloroplast RNA polymerase. Plant nuclear RNA polymerases I, II, and III are much less sensitive to Tagetin Inhibitor. Phage-encoded RNA polymerases such as SP6 and T7 are also relatively insensitive. With both eukaryotic and prokaryotic RNA polymerases, the degree of inhibition is template-dependent. ScriptGuard™ RNase Inhibitor ScriptGuard™ RNase Inhibitor is your best defense against common RNases including RNase A, RNase B, and RNase C. This recombinant RNase inhibitor protein provides reliable protection of your precious RNA samples by binding strongly to RNases in a 1:1 ratio. EPICENTRE’s ScriptGuard™ RNase Inhibitor is free of unwanted contaminants that can plague other commercially available preparations of RNase inhibitors. •A potent affinity for RNases (Ki>10-14 M) ensures rapid inhibition even when trace amounts of RNase are present. •Free of detectable RNase or DNase activity and mammalian DNA. •Does not interfere with enzymes commonly used to prepare or analyze RNA. Catalog No. Conc. Size ScriptGuard™ RNase Inhibitor SRI6325 40 U/µl 2,500 U SRI6310K 40 U/µl 10,000 U •Less sensitive to oxidation than traditional RNase inhibitors. Applications •Effectively inhibits the degradation of RNA by eukaryotic RNases in a variety of applications, including cDNA synthesis, RT-PCR and in vitro transcription and translation. Protein Transport Inhibitor Brefeldin A Brefeldin A (BFA), a metabolite of the fungus Eupenicillium brefeldianum, specifically and reversibly blocks protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in many cell types and species. These effects are generally accompanied by distinct morphological changes, including the apparent collapse of the Golgi stacks. The fast and reversible redistribution of intracellular membranes is accompanied by various specific and reversible effects on cellular protein traffic, including protein transport from the ER to the Golgi, protein secretion, vesicular assembly, antigen presentation, trans- and endocytosis, and viral assembly and budding. Applications •Studying mechanisms of protein transport and targeting. •Inducing “retrograde transport” of proteins normally resident in the Golgi into the ER. •Blocking protein secretion in many cell types. •Blocking antigen presentation by major histocompatibility complex class I and class II molecules. •Reversibly arresting assembly and release of viral particles. Catalog No. Brefeldin A B901MG B905MG Conc. - Size 1 mg 5 mg (5 x 1 mg) •Blocking the toxic effects of ricin, modeccin, abrin, and Pseudomonas toxin in various cell types. •Mapping post-translational modifications of cell-surface receptors and other glycoproteins. FIG 1. Treatment of primary mouse pituitary cells with Brefeldin A. BFA treatment results in the redistribution of Golgi membranes into the endoplasmic reticulum (+BFA) as seen when compared with an untreated cell (-BFA). (Electron micrographs are courtesy of J.A. Magner, Michael Reese Hospital, University of Illinois, Chicago.) Enzyme Storage Buffer Composition Applications 50% glycerol containing 50mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton X-100. • Enzyme storage or dilution. www.EpiBio.com • [email protected] Catalog No. Conc. Enzyme Storage Buffer ESB4901 - Size 1 ml 23 Specialty Enzymes for DNA and RNA Research Contents RNA Polymerases and Replicases RNA Capping and Tailing Enzymes DNA Polymerases Reverse Transcriptase Nucleases and Glycosylases Ligases Phosphatases and Kinase Lysozymes Other Protein products Enzyme Inhibitors page page page page page page page page page page 3 4 6 8 9 18 20 21 22 23 EPICENTRE has the necessary infrastructure, qualified and trained personnel and Quality Systems procedures that are required to manufacture molecular biology enzymes of high purity and quality that will exceed the expectations of the customer. EPICENTRE develops, manufactures and sells optimal enzyme systems and reagents for life science research, diagnostics and pharmaceutical bioprocessing. LARGE QUANTITIES & SPECIAL FORMULATIONS are available. Please call toll free (U.S. only) 1-800-284-8474 to inquire about custom kits or enzyme formulations, highthroughput packaging, bulk orders, animal product-free reagents and manufacturing to meet specific regulatory requirements. EPICENTRE never stops introducing new enzymes for your research needs. Sign up with us at www.EpiBio.com/reply_card.asp to keep updated. USA: 800-284-8474 Tel: 608-258-3080 Fax: 608-258-3088 Technical Support USA: 800-284-8474 [email protected]