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公司介绍
Enzymatics is an emerging leader in the supply of the enzymes that drive nucleic acid detection technologies. Our leadership is currently building a team which embodies our mission to lead the molecular biology tools market in quality, customer servi...

Enzymatics/EnzScript™ (MMLV Reverse Transcriptase RNase H-)/P7600L/10,000 U

Enzymatics/EnzScript™ (MMLV Reverse Transcriptase RNase H-)/P7600L/10,000 U
  • Enzymatics/EnzScript™ (MMLV Reverse Transcriptase RNase H-)/P7600L/10,000 U
商品介绍

EnzScript™(MMLVReverseTranscriptaseRNaseH-)

ProductDescription

EnzScript™(M-MLVReverseTranscriptaseRNaseHminus)isanRNA-dependentDNApolymerasewithnodetectableRNaseHactivity.EnzScript™canbeusedtogeneratefirst-strandCDNAfrompolyAmRNAortotalRNAforuseindownstreamapplicationssuchasRT-PCR,cDNAcloningorlibraryconstructionforRNA-Seq.PointmutationsintheRNaseHdomainincreasetheThermostABIlityoftheenzymeandsupportgreatercDNAyieldoffull-lengthtranscriptsthanwildtypeM-MLVReverseTranscriptase(1).

SourceofProtein:ArecombinantE.colistraincarryingtheMoloney-MurineLeukemiaVirusReverseTranscriptasegenewith3pointmutationsintheRNaseHdomainthateliminatedetectableRNaseHactivity.

UnitDefinition:1unitisdefinedastheamountofenzymerequiredtoincorporate1nmolofdTTPintoacidinsolublematerialin10minutesat37°Cusingpolyr(A)/oligo(dT)asasubstrate.

Molecularweight:75,938Daltons

QualityControlAnalysis

UnitActivity ismeasuredusinga2-foldserialdilutionmethod.Dilutionsofenzymeweremadein1XM-MuLVRTRNAseH-Bufferandaddedto50µLreactionscontaining20µg/mLpolyr(A)RNA,oligo(dT)DNA,1XRTBuffer,3H-dTTPand250µMdTTP.Reactionswereincubated10minutesat37°C,plungedonice,andanalyzedusingthemethodofSambrookandRussell(MolecularCloning,v3,2001,pp.A8.25-A8.26).

ReverseTranscriptasefunction wasdeterminedbytheenzyme’sabilitytogeneratea9.4kBcDNAtranscript.Following2-StepRT-PCRthe9.4Kbampliconwasvisualizedbyagarosegelelectrophoresis

ProteinConcentration(OD280) isdeterminedbyOD280 absorbance.PhysicalPurityisevaluatedbySDS-PAGEofconcentratedanddilutedenzymesolutionsfollowedbysilverstaindetection.Purityisassessedbycomparingtheaggregatemassofcontaminantbandsintheconcentratedsampletothemassoftheproteinofinterestbandinthedilutedsample.

Single-StrandedExonuclease isdeterminedina50µLreactioncontainingarADIolabeledsingle-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°C.

Double-StrandedExonuclease isdeterminedina50µlreactioncontainingaradiolabeleddouble-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°C.

Double-StrandedEndonuclease isdeterminedina50µLreactioncontaining0.5µgofplasmidDNAand10µLofenzymesolutionincubatedfor4hoursat37°C.

E.coli 16SrDNAContamination isevaluatedusing5µLreplicatesamplesofenzymesolutiondenaturedandscreenedinaTaqManqPCRassayforthepresenceofcontaminating E.coli genomicDNAusingoligonucleotideprimerscorrespondingtothe16SrRNAlocus.

Non-SpecificRNAse contaminationisassessedusingtheRNAseAlertkit,(IntegratedDNATechnologies),followingthemanufacturer’sguidelines.

Suppliedin: 20mMTris-HCl,100mMNaCl,0.1mMEDTA,1mMDTT,0.01%NP-40Alternative,50%glycerol,pH7.5@25°C.

Suppliedwith: 5XM-MLVReverseTranscriptaseRNAseH-ReactionBuffer(B7601)and100mMDTT(B9060)

KitContents

PartPartNumber
EnzScript™P7600
5XM-MLVReverseTranscriptaseRNaseH-BufferB7601
100mMDTTB9060

PolymeraseProperties

MolecularWeight:75.9kDa
OptimumExtensionTemperature:42°C
RNaseHactivity:nonedetected
UnitConcentration:200U/µl
TranscriptLength:12.3kB

CommonApplications

EnzScript™(M-MLVReverseTranscriptaseRNaseHminus)isanRNA-dependentDNApolymerasecommonlyusedtosynthesizeFirst-StrandcDNAforRT-PCRamplification,cDNAcloningorRNASeq.ReducedRNaseHactivityenablesgreateryieldoffull-lengthcDNAtranscripts(>5kb)andincreasedthermalstabilityoverstandardM-MLVRT.

FirstStrandReactionProtocol

GeneralprecautionagainstRNasedegradationoftemplateRNAshouldbetakenwhensettingupFirst-Strandreactionssuchasuseofnuclease-freewater,RNaseinhibitor,RNase-freetubesandsterilepipettipswithfilters.Thefollowingprocedurecanbeusedasaguidelineforpreparinga20µlFirst-StrandcDNAreaction.

MaterialstobeprovidedbyUser:

•Sterile,nuclease-freewater
•Primer(oligodT(15-20) or randomhexamers or gene-specific)
•dNTPmix(dATP,dCTP,dGTP,dTTP)
•RNAtemplate
•RNaseInhibitor

1.AddthefollowingcomponentstoanRNase-freemicrocentrifugetubeonice.Formorethanonereaction,prepareamastermix.

ComponentVolume
50µMoligodT(15-20)or
50µMrandomhexamersor
10µMgene-specificprimer
1to2µl
5mMdNTPmix2µl
RNAtemplate*Xµl
Sterile,nuclease-freewaterto12µl

*1ngto1µgtotalRNA or 1to250ngmRNA

2.Heatmicrocentrifugetubeto65°Cfor5minutesandquicklycoolonicefor2minutestoannealprimertoRNAtemplate.Spintubebrieflytocollectcondensate.

3.Addthefollowingcomponents(toeachFirst-Strandreaction)tothemicrocentrifugetubeonice:

ComponentVolume
5XM-MLVReverseTranscriptase
RNaseH-Buffer
4µl
100mMDTT2µl
RNaseInhibitor(optional)or
nuclease-freewater
1µl
200UEnzScript™1µl

4.Incubateeach20µlFirstStrandreactionasfollows:

  1. 25°Cfor2minutes(oligodT(15-20),gene-specificprimer) or 25°Cfor10minutes(randomhexamer)
  2. 42°Cfor30to60minutes
  3. Heatkillat70°Cfor15minutes

5.UsecDNAindownstreamapplicationorstoreat-20°C.ForRT-PCR,1to2µlofcDNAfromFirst-StrandreactionistypicallyaddedastemplatetoPCR.Optional:RemoveRNAstrandpriortoPCRbyadding1µlRNaseH(5U)tocDNA:RNAhybrid,incubateat37°Cfor20minutesand65°Cfor10minutes(heatkill).RNaseHtreatmentisrecommendedforamplificationoflongamplicons(>5kB).

RelatedEnzymaticsProducts

PartPartNumber
25mMdNTPmixN2050
RNaseInhibitorY9220
RNaseHY9240

FrequentlyAskedQuestionsandTroubleshooting

ForFrequentlyAskedQuestions(FAQ)andtroubleshootingpleaseclickhere.

ProductInformation

EnzScript™(MMLVReverseTranscriptaseRNaseH-)
PartNumberP7600L
Price$128/pack
Concentration200,000U/mL
UnitSize10,000U
LotNumberShipmentdependent
SDSAvailableonrequest

ProductSpecification*

StorageTemperature-25°Cto-15°C
TestUnitsTestedSpecification
SDSPurityn/a>99%
SpecificActivityn/a≥280,000U/mg
SSExonuclease2,000<5.0%
DSExonuclease2,000<1.0%
DSEndonuclease2,000NoConversion
E.coliDNAContamination2,000<10copies
RNAseContamination2,000NoDetectablenon-specificRNase
FunctionalRT-PCRAssayn/aSynthesisof9.4kbcDNAtranscript

*Foradetailedsummaryofassayconditionsanddata,refertotheQualityControlsAnalysissection.

AdditionalDownloads

References

  1. GerardG.F.etal.,Nucl.AcidsRes.(2002)30(14):3118-3129

LimitationsofUse
Thisproductwasdeveloped,manufactured,andsoldforinvitrouseonly.TheproductisnotsuitableforadmiNISTrationtohumansoranimals.SDSsheetsrelevanttothisproductareavailableuponrequest.

品牌介绍

Enzymatics is an emerging leader in the supply of the enzymes that drive nucleic acid detection technologies. Our leadership is currently building a team which embodies our mission to lead the molecular biology tools market in quality, customer service, and innovation. We seek engaging, motivated individuals who can articulate their goals and, in turn, offer a culture that breeds success. Enzymatics is an employee-focused organization that strives to cultivate challenging careers and rewarding lives. If you seek a challenge, have a thirst for knowledge, and are interested in having fun while working, please apply to learn more!

We offer a competitive benefits package and an incentive-based compensation package.



苏州蚂蚁淘生物科技有限公司是一家创新型高科技生物公司,以常规生物学实验外包与生物学试剂为基础,放眼于生命科学领域的前沿技术服务,公司技术骨干有着多年的生物学科研背景,毕业于国内外名牌大学,并且有着丰富的项目经验,了解国内外科研行情,我公司力求为每一个客户提专业、针对性的实验方案。我公司以技术服务为支点,和国内外多家生物试剂公司有着密切的业务往来.公司特色技术服务包括:抗体药物研发、噬菌体抗体库、蛋白表达与传化、抗体制备、高通量测序、组学研究、生物信息学分析与方法建立、基础分子生物学技术外包、化学中间体合成、原料药物研发、论文编写等。我公司秉承”诚信做人,认真做事,服务至上“,希望用我们的专业知识和项目经验为国内科研客户解决科研问题,祝您科研之路畅通无阻!


Enzymatics是马萨诸塞州贝弗利市一家快速发展的生物技术公司,已成为推动核酸检测技术的酶供应的者。

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10× Fragmentation Buffer

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无海藻糖5X一步法反转定量Buffer

 

93.6

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B0340L

Enhancer buffer

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B1010L

2×Rapid Ligation Buffer

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2×Rapid Ligation Buffer

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2X 快速连接缓冲液

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10×T4 DNA Ligase Buffer

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10X T4 DNA 连接酶缓冲液200 ml

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B6060L-40

10×Taq连接酶反应缓冲液

40 ml

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B6090L-40

10 × E.coli DNA Ligation Buffer

40 ml/瓶

3848

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B7020L-40

10 × φ29 DNA Polymerase Reaction Buffer

40 ml/瓶

3848

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B7030

10×PCR Buffer I

1 ml

96.2

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B7030L-50

10 × PCR Buffer I

50 ml/瓶

4810

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10 × M MuLV RT Reaction Buffer

40 ml/瓶

3848

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B7102L-40

5 × VS Buffer 2

40 ml/瓶

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5 × GC Buffer

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