T7,T3,andSP6RNAPolymerasesThe best value in phage RNA polymerases.
• High purity, promoter specificity, and activity.• Free of detectable DNase and RNase activities.• Function-tested in in vitro transcription reactions.
All RNA Polymerases are available in bulk. T7 and T3 RNA Polymerases are also available at concentrations up to 2,500 Units/µl. Please contact EPICENTRE for custom orders.
Cat. # Quantity
E. coli RNA Polymerase Core Enzyme C90100 100 Units C90500 500 Units
Sigma-Saturated E. coli RNA Polymerase HoloenzymeS90050 50 Units S90250 250 Units
E. coliRNAPolymerases(CoreandHoloenzyme)EPICENTRE is the only commercial source for both:
• Purified Core Enzyme that has no detectable sigma subunit by gel electrophoresis.• Purified Holoenzyme that is 100% sigma-saturated for efficient transcription of DNA templates
containing promoters.Perform rapid, high throughput screening of bacterial RNA polymerases and inhibitors using the Kool™ NC-45 RNAP Activity and Inhibitor Screening Kit. Visit www.EpiBio.com for details.
Figure 1. SubunitpatternsofE. coliRNAPolymeraseCoreEnzymeandHoloenzymepreparations. 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 below detectable limits in the Core Enzyme.
Holo Core
– β′, β– σ
– α
– ω
Cat. # Concentration Quantity
T7 RNA Polymerase T7905K 50 U/µl 5,000 Units T7950K 50 U/µl 50,000 Units TM910K 200 U/µl 10,000 Units (Enzyme only. Transcription Buffer not included.)
T3 RNA Polymerase T4905K 50 U/µl 5,000 Units T9050K 50 U/µl 50,000 Units (Enzyme only. Transcription Buffer not included.)
SP6 RNA Polymerase S4905K 50 U/µl 5,000 Units S4950K 50 U/µl 50,000 Units SM910K 200 U/µl 10,000 Units (Enzyme only. Transcription Buffer not included.)
Transcription Buffer Package BP1001 1 Pkg Includes 5 ml of 5X Transcription Buffer and 2.5 ml of 100 mM DTT.
T7R&DNA™PolymeraseT7 R&DNA™ Polymerase is a mutant form of the wild-type T7 RNA Polymerase that:
• Readily incorporates 2′-dNTPs and other 2′-modified-dNTPs, as well as canonical NTPs. • Produces “RNA” of mixed rNMP/2′-dNMP or rNMP/2′-modified-NMP composition. • Produces modified “RNA” transcripts that are resistant to RNase A and related RNases.• Uses the same transcription promoters as the wild-type T7 RNA polymerases.
The DuraScribe® T7 Transcription Kit for making 2′-fluorine-modified RNA that is completely resistant to RNase A is also available (see p. 8).
Cat. # Concentration Quantity
T7 R&DNA™ PolymeraseD7P9201K 50 U/µl 1,000 Units D7P9205K 50 U/µl 5,000 Units Includes 5X Reaction Buffer, DTT.
Cat. # Concentration Quantity
ATP R109AT 10 mM 5 µmol RA02825 100 mM 25 µmol
CTP R109CT 10 mM 5 µmol RC02825 100 mM 25 µmol
GTP R109GT 10 mM 5 µmol RG02825 100 mM 25 µmol
UTP R109UT 10 mM 5 µmol RU02825 100 mM 25 µmol
One Premixed Solution of all 4 NTPs R344NT 2.5 mM each 5 µmol each (Premix in one tube contains ATP, CTP, GTP & UTP)
One Tube of each NTP (ATP, CTP, GTP, UTP)RN02825 100 mM each 25 µmol each (Not premixed)
2′-Fluorine-dCTP (2′-F-dCTP) R2F110C 50 mM 1 µmol
2′-Fluorine-dUTP (2′-F-dUTP) R2F110U 50 mM 1 µmol
Aminoallyl-UTP AAU5202 50 mM 2.5 µmol
Biotin-16-UTPBU6105H 50 mM 500 nmol
NTPsEPICENTRE’s NTPs are supplied as sterile, neutral aqueous solutions.
• Are rapid (30-minute) reactions that produce more RNA than other kits produce in 2 hours. • Transcribe 180 µg of RNA from 1 µg of DNA template.• Produce high yields of RNA from both long and short DNA templates.• Generate high yields of RNA even from limiting amounts of DNA template.
Cat. # Quantity
AmpliScribe™ T7-Flash™ Transcription KitASF3257 25 Reactions ASF3507 50 Reactions Contents: AmpliScribe™ T7-Flash™ Enzyme Solution (with added RNase Inhibitor), AmpliScribe™ T7-Flash™ 10X Reaction Buffer, 100-mM ATP, CTP, GTP and UTP Solutions, RNase-Free Water, RNase-Free DNase I, DTT, Control DNA Template (linearized).
AmpliScribe™ T3 High Yield Transcription KitAS3103 50 Reactions
AmpliScribe™ SP6 High Yield Transcription KitAS3106 50 Reactions Contents: AmpliScribe™ T7, T3, or SP6 Enzyme Solution (with added RNase inhibitor), 100-mM ATP, CTP, GTP, and UTP Solutions, AmpliScribe™ 10X Reaction Buffer, RNase-Free Water, RNase-Free DNase I, DTT, Control Template DNA (linearized).
AmpliScribe™T7,T3,andSP6HighYieldTranscriptionKits• Produce up to 150 µg of RNA in a 2-hour reaction.
Figure 1. AnAmpliScribe™T7-Flash™transcriptionreactioniscompletein30minutesandyieldsmoreRNAthanother2-hour"high-yield"transcriptionreactions.Yields shown were obtained using 1 µg of a linear DNA template in a 20-µl reaction, producing a 1.4-kb RNA.
AmpliScribe™T7-Flash™Biotin-RNATranscriptionKitThe AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit enables high-yield synthesis of biotin-RNAor biotin-antisense RNA (aRNA, also called cRNA) for use as microarray target or as probe for in situ or blotting experiments.
• The kit uses EPICENTRE’s AmpliScribe T7-Flash rapid, high-yield in vitro transcription technology to generate the highest possible yield of biotin-RNA.
• Reaction conditions are optimized to yield uniformly labeled biotin-RNA for high signal intensity.
Cat. # Concentration Quantity
AmpliScribe™ T7 Aminoallyl-RNA Transcription KitAA50125 25 Reactions Contents: AmpliScribe™ T7 Aminoallyl-RNA Enzyme Solution (with added RNase Inhibitor), AmpliScribe™ T7 Aminoallyl-RNA 10X Reaction Buffer, ATP, CTP, GTP, UTP and Aminoallyl-UTP Solutions, RNase-Free Water, RNase-Free DNase I, DTT, Control DNA Template (linearized).
AmpliScribe™ T7-Flash™ Biotin-RNA Transcription KitASB71110 10 Reactions Contents: AmpliScribe™ T7-Flash™ Enzyme Solution, ScriptGuard™ RNase Inhibitor, AmpliScribe™ T7-Flash™ 10X Reaction Buffer, NTP/Biotin-UTP PreMix (ATP, CTP, GTP, UTP, and Biotin-16-UTP) Solution, RNase-Free Water, RNase-Free DNase I, DTT, Control DNA Template (linearized).
AmpliScribe™T7Aminoallyl-RNATranscriptionKitThe AmpliScribe™ T7 Aminoallyl-RNA Transcription Kit incorporates Aminoallyl-UTP into RNA transcripts to generate high yields of aminoallyl-RNA (AA-RNA). The AA-RNA produced can be subsequently labeled and used as microarray target or as probe for in situ hybridization or blotting experiments.
• High yields of Aminoallyl-RNA (over 150 µg of RNA from 1 µg of DNA template).• Reaction conditions are optimized to yield uniformly labeled aminoallyl-RNA for high signal
intensity following dye or biotin conjugation. • Less expensive than direct incorporation of fluorescent- or biotin-labeled NTP.
AmpliCap™HighYieldMessageMakerKitsThe AmpliCap-Max™ and AmpliCap™ Kits are formulated to produce exceptionally high yields of 5′-capped RNA for use in in vitro translation, microinjection, and mRNA processing studies.
• Kits incorporate m7GpppG cap analog at the 5′ end of the transcribed RNA.• AmpliCap-Max™ T7 & T3 Kit reactions yield up to 60 µg of RNA from 1 µg of template.• Capping efficiencies of up to 80%.• Optimized m7GpppG/NTP PreMix is provided.• AmpliCap-Max™ T7 and T3 Kit reactions are complete in 30 minutes.
Cat. # Quantity
AmpliCap-Max™ T7 High Yield Message Maker KitACM04037 25 Reactions
AmpliCap-Max™ T3 High Yield Message Maker KitACM04033 25 Reactions Contents: AmpliCap-Max™ T7 or T3 Enzyme Solution (with added RNase Inhibitor), AmpliCap-Max™ Cap/NTP PreMix, 20 mM GTP, AmpliCap-Max™ 10X Transcription Buffer, RNase-Free Water, RNase-Free DNase I, DTT, Control Template DNA (linearized).
AmpliCap™ SP6 High Yield Message Maker KitAC0706 25 Reactions Contents: AmpliCap™ SP6 Enzyme Solution (with added RNase Inhibitor), AmpliCap™ 10X Transcription Buffer, AmpliCap™ Cap/NTP PreMix, 20 mM GTP, RNase-Free DNase I, DTT, RNase-Free Water, Control Template DNA (linearized).
Cat. # Quantity
Standard Cap Analog, m7G[5′]ppp[5′]G SolutionC61025 1,250 nmol
Unmethylated Cap Analog, G[5′]ppp[5′]G SolutionC32010 500 nmol
Trimethylated Cap Analog, m32,2,7G[5′]ppp[5′]G Solution
C06005 250 nmol
CapAnalogsEach Cap Analog is:
• Function tested as a substrate for capping an RNA transcript synthesized in an in vitro transcription reaction.
Poly(A)PolymeraseTailingKitEPICENTRE’s Poly(A) Polymerase is a cloned enzyme that uses ATP for template-independent addition of adenosine nucleotides to the 3′-hydroxyl terminus of RNA molecules.
Figure 1. Poly(A)tailingofRNAwiththePoly(A)PolymeraseTailingKit.The entire final product of an AmpliCap-Max™ in vitro transcription capping reaction (60 µg of a 1,760-base luciferase RNA) was treated with 8 units of Poly(A) Polymerase for the indicated times. For each sample, 0.1-µg aliquots were analyzed on a 1% agarose-formaldehyde gel.
Add a 3′-poly(A) tail to your RNA to:• Increase the stability and enhance the translation of an RNA. • Provide an oligo(dT) priming site to poly(A)− RNAs, such as miRNA or bacterial RNAs.• 3′-end label RNA or miRNA.
DuraScribe®T7&SP6TranscriptionKitsThe DuraScribe® T7 and SP6 Transcription Kits* produce 2′-Fluorine-modified RNA transcripts—called DuraScript® RNA—that are completely resistant to RNase A. DuraScribe T7 and SP6 RNA Polymerases are mutant forms of T7 and SP6 RNA Polymerases that efficiently incorporate 2′-Fluorine-dCTP (2′-F-dCTP) and 2′-Fluorine-dUTP (2′-F-dUTP), as well as ATP and GTP into full-length transcripts. They use the same promoters as wild-type T7 and SP6 RNA polymerases. The presence of the 2′-F-dC and 2′-F-dU nucleotides render DuraScript RNA completely resistant to RNase A and related ribonucleases. A standard DuraScribe T7 reaction produces about 50 µg of DuraScript RNA.
Use DuraScript RNA for:
• RNAi, ribozyme, and antisense RNA studies.• In situ hybridization.• Ribonuclease protection assays.
*Covered by issued and/or pending patents. See www.EpiBio.com/legal.
Figure 2. DuraScript®RNAisresistanttoRNaseAdigestion.A 1.4-kb standard RNA transcript and a 1.4-kb DuraScript RNA transcript were each incubated with 1 U of highly purified RNase A for 30 minutes. The standard RNA transcript was completely degraded while the DuraScript RNA transcript remained intact. Lane M, Size ladder; lane 1, 1.4-kb standard RNA transcript; lane 2, standard RNA after RNase A treatment; lane 3, 1.4-kb DuraScript RNA; lane 4, DuraScript RNA after RNase A treatment.
ScriptGuard™RNaseInhibitorEPICENTRE’s recombinant ScriptGuard™ RNase Inhibitor provides reliable protection for valuable RNA samples by binding strongly to RNase A–type ribonucleases in a 1:1 ratio.
• 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.• Less sensitive to oxidation than traditional RNase inhibitors.
Cat. # Quantity
ScriptGuard™ RNase InhibitorSRI6325 2,500 Units SRI6310K 10,000 Units
Cat. # Concentration Quantity
Thermostable RNA LigaseTRL8101K 50 U/µl 1,000 Units
T4 RNA Ligase is also availableLR5010 5 U/µl 1,000 Units LR5025 5 U/µl 2,500 Units
ThermostableRNALigaseThermostable RNA Ligase catalyzes the intra- and intermolecular ligation of single-stranded RNA (ssRNA) molecules that have a 5′-monophosphate and a 3′-hydroxyl end. The enzyme can be used for:
• Ligation-tagging of 5′-monophosphorylated RNA.• Generating 5′-tagged RNA for RACE or other application.• Circularization of RNA molecules.
RNASE Inhibitor and RNA Modifying Enzymes
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TobaccoAcidPyrophosphataseTobacco Acid Pyrophosphatase (TAP) efficiently removes the cap structure present at the 5′ end of most eukaryotic mRNAs, viral RNAs, and small capped RNAs. The end-product of a TAP reaction is a 5′-phosphorylated RNA. TAP can be used to:
• Prepare 5′-capped RNAs for 5′-ligation tagging reactions.• Prepare template for 5′-RACE.• Characterize the 5′-end structure of RNAs.
RNA5’PolyphosphataseRNA 5′ Polyphosphatase,* discovered and characterized by EPICENTRE scientists, sequentially removes the γ- and β-phosphates from 5′-triphosphorylated RNA (such as uncapped primary RNA transcripts):
5′pppN—OH3′➞5′pN—OH3′+2Pi
Unlike Tobacco Acid Pyrophosphatase, RNA 5′ Polyphosphatase has no activity on RNA with a 5′ cap (e.g., 5′ m7GpppN—OH 3′). RNA 5′ Polyphosphatase can be used to:
• Prepare uncapped primary transcripts for 5′-ligation-tagging reactions.• Prepare template from uncapped primary transcripts for 5′-RACE.• Characterize the 5′ terminus of RNA molecules.
*Covered by issued and/or pending patents. See www.EpiBio.com/legal.
RiboShredder™RNaseBlendRiboShredder™ RNase Blend is a cocktail of potent, nonmammalian RNases that completely degrades RNA.
• Complete removal of RNA from genomic DNA preparations.• Completely degrades RNA to mononucleotides.
Cat. # Concentration Quantity
RiboShredder™ RNase BlendRS12100 1 U/µl 100 Units RS12500 1 U/µl 500 Units
Cat. # Concentration Quantity
E. coli RNase HR52250 10 U/µl 250 Units R0601K 10 U/µl 1,000 Units
AdditionalRNasesfromEPICENTRE
Hybridase™ Thermostable RNase H, RNase T1, RNase R, RNase A, RNase I (E. coli) and RNase III (E. coli). Please visit www.EpiBio.com for details.
E. coli RNase HE. coli RNase H specifically degrades the RNA in a DNA:RNA hybrid without affecting dsDNA, ssDNA, dsRNA, or unhybridized RNA. E. coli RNase H is commonly used to synthesize ds cDNA from RNA.
Figure 1. ComparisonofRNA-degradingcapabilityofRiboShredder™RNaseBlendversuscommonly-usedindividualRNasesorotherRNasecocktails.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 M, supercoiled DNA ladder.
1 2 3 4 5 6 M
RiboShredder™ RNase Blend➞
Ribonucleases
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Terminator™5’-Phosphate-DependentExonucleaseTerminator™ Exonuclease is a 5′→3′ processive exonuclease that degrades RNAs with a 5′ monophosphate, such as large rRNAs. RNAs with a 5′-cap structure, such as eukaryotic mRNA, or a 5′ triphosphate, such as prokaryotic mRNAs, are resistant to Terminator Exonuclease digestion.
• Selectively digests prokaryotic 16S and 23S rRNAs, and eukaryotic 18S and 28S rRNAs, in a total RNA preparation.
• A simple 1-hour enzymatic reaction enriches for prokaryotic or eukaryotic mRNA without the need for columns, resins, or beads.
• Not inhibited by commonly used RNase inhibitors.
Cat. # Concentration Quantity
Terminator™ 5′-Phosphate-Dependent ExonucleaseTER51020 1 U/µl 20 Units (1 Unit enriches mRNA from up to 10 µg of total RNA)Includes Terminator™ Exonuclease 10X Reaction Buffer.
MonsterScript™ReverseTranscriptaseMonsterScript™1st-StrandcDNASynthesisKit• Capable of producing full-length cDNA greater than 15 kb.• Thermostable: retains high activity to >50°C. • Completely lacks RNase H activity (RNase H−). • Efficiently produces cDNA from picogram amounts of total RNA.• The MonsterScript™ 1st-Strand cDNA Synthesis Kit includes a convenient PreMix containing
betaine* to reduce pausing and stops observed with other reverse transcriptase enzymes.
*Covered by issued and/or pending patents. See www.EpiBio.com/legal.
MonsterScript™ 1st-Strand cDNA Synthesis KitMS040910 10 Reactions MS041050 50 Reactions Contents: MonsterScript™ Reverse Transcriptase (50 U/µl) with RNase Inhibitor, MonsterScript™ 5X cDNA PreMix (contains buffer, dNTPs, and betaine*), V3-Oligo(dT)21 Primer (10 µM), Random 9-mer Primers, RNase-Free Water.
Figure 1. MonsterScript™ReverseTranscriptaseproducesfull-lengthcDNAfrommRNAgreaterthan15kb.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 (A). Agarose gel electrophoresis of the 1.3-kb amplicon from the 5′ end of the mRNA demonstrates full-length cDNA synthesis (B).
A ~ 15.2 kb HGNEFp532 mRNA
5’ AAAAA . . . -3’
1.3 kbPCR i4
7040
7ms
B
kb
1.6—
1.0—
cDNA Synthesis
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CircLigase™IIssDNALigaseCircLigase™ II ssDNA Ligase* is a thermostable enzyme that catalyzes circularization of single-stranded DNA (ssDNA) templates, including cDNA, having a 5′-phosphate and a 3′-hydroxyl group. The enzyme circularizes ssDNA molecules >15 bases, including cDNAs, without the need for oligo “bridges,” “splints” or T4 DNA Ligase. Under standard conditions, virtually no linear or circular concatamers are produced.
CircLigase™ II ssDNA Ligase can be used for:
• Production of ssDNA templates for rolling-circle replication or rolling-circle transcription experiments.
• Production of ssDNA templates for RNA polymerase and RNA polymerase inhibitor assays.
The enzyme offers the following benefits:• It circularizes ssDNA molecules >15 bases, including cDNAs, without the need for oligo bridges or
splints.• Virtually no linear or circular concatamers are produced under standard reaction conditions.
*Covered by issued and/or pending patents. See www.EpiBio.com/legal.
Cat. # Concentration Quantity
CircLigase™ II ssDNA LigaseCL9021K 100 U/µl 1,000 Units CL9025K 100 U/µl 5,000 Units
Figure 1. CircLigase™IIssDNALigaseconvertslinearssDNAintoclosed-circularssDNA.A 71-base ssDNA oligo was converted to a circular DNA form in a reaction containing MnCl2 and CircLigase II ssDNA Ligase. Lane 1, DNA markers; lane 2, 71-base linear ssDNA oligo; lane 3, circularization proceeds through an adenylated intermediate; lane 4, closed-circular ssDNA reaction product.
Exo I & III + +
CircLigase™ II Ligase − + M
bp
12,216—
506—
220—
Figure 2. PreparationofacircularssDNAlibrary.5′-end-tagged and fragmented T7 D111 genomic DNA was heat-denatured and then circularized by incubating in 33 mM Tris-acetate pH 7.6, 66 mM KOAc, 2.5 MnCl2, and 1 M betaine for 2 hours at 60°C in the presence or absence of 400 U of CircLigase™ II enzyme, as indicated. Reaction products were incubated with exonuclease I and exonuclease III to digest linear DNA prior to PCR amplification with primers complementary to each tag. Reaction products were visualized by SYBR® Gold staining.
The TargetAmp™ RNA Amplification Kits are the most robust kits for producing labeled target aRNA (also called cRNA) from small samples for transcription profiling or expression microarray studies.
Features include:
• High yield/low input of RNA. • Fast reactions.• Linear RNA amplification process.• High-quality microarray results on Affymetrix® GeneChip®, Illumina® BeadArray and other
commercial and spotted arrays. • Evaluation kit program is available.
TargetAmp™ 1-Round RNA Amplification Kit reactions are single-tube reactions that can be completed in about 6 hours. The TargetAmp 1-Round Kit reactions produce microgram quantities of aRNA from 25 to 500 ng of input total RNA.
TargetAmp™ 2-Round RNA Amplification Kits are the only RNA amplification kits that enable microarray studies from a single cell. The TargetAmp 2-Round Kit reactions produce microgram quantities of aRNA from 10 to 500 pg of input total RNA.
The MessageBOOSTER™ cDNA Synthesis Kits amplify the poly(A) RNA contained in a total RNA preparation and then convert the amplified RNA to cDNA that is ready for PCR or qPCR.
• Reproducibly detect even low-abundance transcripts from as little as one cell.• Get hundreds of qRT-PCRs from as little as one cell.• Collect samples less often and archive valuable samples. • One-day reaction. • Linear RNA amplification process preserves the relative abundance of transcripts present in the
sample.
Three MessageBOOSTER Kits are available that enable the researcher to work with purified total RNA, compromised or partially degraded total RNA, or to perform a MessageBOOSTER cDNA synthesis reaction directly from a cultured-cell lysate without having to purify RNA.
RiboMultiplier™Sense-RNAAmplificationKit
The RiboMultiplier™ Kit utilizes a unique terminal tagging process to produce high yields of amplified sense RNA (sRNA).
• Produce micrograms of sense RNA from as little as 10 ng of input total RNA.• Sense RNA produced has excellent 3′/5′ ratio.• Sense RNA produced demonstrates excellent correlation on microarrays with MAQC TaqMan® data.
Visit www.Epibio.com/RNA_Amp for additional information, and pricing for all RNA amplification kits.
