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当前位置: 首页 > 产品中心 > DNA Modifying Enzymes > Enzymatics/10x Uracil Cleavage System/Y9180L/750 Reactions
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/10x Uracil Cleavage System/Y9180L/750 Reactions

Enzymatics/10x Uracil Cleavage System/Y9180L/750 Reactions
  • Enzymatics/10x Uracil Cleavage System/Y9180L/750 Reactions
商品介绍

10xUracilCleavageSystem

ProductDescription

TheUracilCleavageSystemprovidestwoenzymes,which,whenaddedsequentiallytoareactioncontainingasyntheticDNAfragmentcontainingadeoxy-uracil,generateasinglenucleotidegapatthelocationoftheuracilresidue.Thesystemconsistsoftwoindividualenzymecomponents,UracilDNAGlycosylase(UDG)andEndonucleaseVIII,providedata10Xconcentrationtobeaddedtoareactioncontainingauracil-containingpolynucleotidesequence.UDGcatalysestheexcisionoftheuracilbase,creatinganabasicsitewithanintactphosphodiesterbackbone(1,2).ThelyaseactivityofEndonucleaseVIIIbreaksthephosphodiesterbackboneboth3′and5′totheabasicsite,liberatingthedeoxyribosesugar(3,4).

SourceofProtein
Eachcomponentproteinispurifiedseparatelyfrom E.colistrainscontainingrecombinantEndonucleaseVIIIandUracil-DNAGlycosylasegenes.

Suppliedwith:
UDG(G5010)

10mMTris-HCl
50mMNaCl
1mMDTT
0.1mMEDTA
50%glycerol
pH7.5@25°C

EndonucleaseVIII(Y9080):
10mMTris-HCl
250mMNaCl
0.1mMEDTA
50%glycerol
pH8.0@25°C

UnitDefinition,UDG
1unitisdefinedastheamountofenzymethatcatalyzesthereleaseof1.8nmolofuracilin30minutesfromdouble-stranded,tritiated,uracilcontaining-DNAat37°Cin1XUDGReactionBuffer.

UnitDefinition,EndonucleaseVlll
Oneunitisdefinedastheamountofenzymerequiredtocleave1pmolofanoligonucleotideduplexcontainingasingleAPsitein1hourat37°C.

QualityControlAnalysis

UnitCharacterizationAssay,UDG
Specificactivitywasmeasuredusinga2-foldserialdilutionmethod.Dilutionsofenzymeweremadein1Xreactionbufferandaddedto50µLreactionscontaininga3H-dUTPcontainingPCRproductand1XUDGReactionBuffer.Reactionswereincubatedfor10minutesat37°C,plungedonice,andanalyzedusingaTCA-precipitationmethod.

UnitCharacterizationAssay,EndonucleaseVIII
Specificactivitywasmeasuredusinga2-foldserialdilutionmethod.Dilutionsofenzymeweremadein1Xreactionbufferandaddedto10µLreactionscontaining2.0µMofaFAM-labeled,34-baseduplexoligonucleotide,containingasingleUracil.[Note:substratepre-treatedfor2minuteswithUDGtocreateanabasicsite]Reactionswereincubated60minutesat37°C,plungedonice,denaturedwithN-N-dimethyl-formamideandanalyzedona15%TBE-Ureaacrylamidegel.

SDS-Page(PhysicalPurityAssessment)
2.0µLofenzymesolutionwasloadedonadenaturing4-20%Tris-GlycineSDS-PAGEgelflankedbyabroad-rangeMWMarkerand2.0µLofa1:100dilutionofthesample.Followingelectrophoresis,thegelwasstainedandthesamplescomparedtodeterminephysicalpurity.Theacceptancecriteriaforthistestrequiresthattheaggregatemassofcontaminantbandsintheconcentratedsampledonotexceedthemassoftheproteinofinterestbandinthedilutesample,confirminggreaterthan99%purityoftheconcentratedsample.

ContaminationTests

(PerformedonUDGandEndonucleaseVIIIseparately):

Single-StrandedExonucleaseActivity
A50µLreactioncontaining10,000cpmofarADIolabeledsingle-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°Cresultedinlessthan5.0%releaseofTCA-solublecounts.

Double-StrandedExonucleaseActivity
A50µLreactioncontaining5,000cpmofaradiolabeleddouble-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°Cresultedinlessthan1.0%releaseofTCA-solublecounts.

Double-StrandedEndonucleaseActivity
A50µLreactioncontaining0.5µgofpBR322DNAand10µLofenzymesolutionincubatedfor4hoursat37°CresultedinnovisuallydiscernIBLeconversiontonickedcircularDNAasdeterminedbyagarosegelelectrophoresis.

E.coli16SrDNAContaminationTest
Replicate5µLsamplesofenzymesolutionweredenaturedandscreenedinaTaqManqPCRassayforthepresenceofcontaminatingE.coligenomicDNAusingoligonucleotideprimerscorrespondingtothe16SrRNAlocus.Theacceptancecriterionforthetestisthethresholdcyclecount(Ct)producedbytheaverageof3replicatenotemplatecontrolsamples.BasedonthecorrelationbetweenthenotemplatecontrolCtvalues,andstandardcurvedata,thedetectionlimitofthisassayis<10copiesgenome/sample.

ProductInformation

10xUracilCleavageSystem
PartNumberY9180L
Price$363
ConcentrationN/A
UnitSize750Reactions
SDSAvailableonrequest

ProductSpecification*

StorageTemperature-25to-15°C
TestSpecification
Purity(SDS-PAGE)>99%
SSExonuclease10µL<5.0%released
DSExonuclease10µL<1.0%released
DSEndonuclease10µL=Noconversion
E.coliDNAContamination5µL<10%copies

*Foradetailedsummaryofassayconditionsanddata,refertotheQualityControlsAnalysissection.

UsageInstructions

Thefollowingreactionspecificationissetto10µl.Thevolumecanbetailoredupordowntosuittherequirementoftheapplicationbymaintainingtheratiosofcomponentsoutlinedbelow.

  1. PrepareUracil-containingDNA(e.g.PCRamplificationproduct).TheUracilCleavageSystemenzymesareactiveinmostmolecularBIOLOGyreactionbuffers,sothereisnoneedtoexchangebufferspriortoassemblingthecleavagereaction.
  2. Combineandmixthefollowingcomponentsinasteriletube:
    AmountDescriptionFinalConcentration
    1-10µLUracil-containingDNA(upto3pmol)Upto300nM
    to10µLTypeIWaterN/A
    10µLTotalVolume
  3. Add0.5µlUDG(G5010)toareactionvesselcontainingtheDNAsubstrate.
  4. Add0.5µlEndonucleaseVIII(Y9080)toareactionvesselcontainingtheDNAsubstrate.
  5. Incubateat37°Cfor30minutes.
  6. Followingtheincubationperiod,thereactioncanbestoppedbyaddingagel-loadingstopsolutioninpreparationforelectrophoreticanalysis,orpreparedfortransformation.Inthecaseoftransformation,thefragmentmaybeligatedintoacloningvectorusingT4DNALigase(PartNumber:L6030-HC-L)

References

  1. Lindhal,T.etal.(1977)J.Biol.Chem.,252,3286-3294.
  2. Lindhal,T.(1982)Annu.Rev.Biochem.,51,61-64.
  3. Melamede,R.J.etal.(1994)Biochemistry,33,1255-1264.
  4. Jiang,D.etal.(1997)J.Biol.Chem.,272,32230-32239.

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!


货号:Enzymatics Y9040L

品名:T4 Polynucleotide Kinase(T4多核苷酸激酶)

产品描述:

T4多核苷酸激酶(PNK)催化ATP的末端γ位磷酸酯转移和交换到双链和单链DNA,RNA和核苷3-单磷酸酯分子的5-羟基末端(1)。T4 PNK还表现出3-磷酸酶和2,3环磷酸二酯酶活性(2,3,4,5,6)。

 

规格:

10,000 U

浓度

10,000 U/ml

保存

-25°C至-15°C

纯度(SDS-PAGE)

> 99%

活性:

133,333U / mg

SS核酸外切酶

2000 U <5.0% released

DS核酸外切酶

2000 U <1.0% released

DS内切核酸酶

2000 U = No conversion

大肠杆菌 DNA污染

2000 U <10 copies

 

蛋白质来源

从表达重组T4多核苷酸激酶基因的大肠杆菌中纯化而得。

 

补充:

10 mM Tris-HCl

50 mM KCl

0.1 碌M ATP

1.0 mM DTT

0.1 mM EDTA

50% glycerol

pH 7.4 @ 25掳C

 

随附:

B9040 (10X T4 Polynucleotide Kinase Buffer):

 

10X多核苷酸激酶缓冲液(B9040)700mM

Tris-HCl 

100mM MgCl 2

50mM DTT 

pH 7.6在25℃

 

单位定义
一个单位定义为在1X T4多核苷酸激酶反应缓冲液中,37℃下30分钟内催化掺入1 nmol [ 32 P](ATP供体)的酶量。

 

质量控制分析

 

单位表征测定
单位活性使用2倍系列稀释法测量。在1X反应缓冲液中制备酶的稀释液,并将其加入到50μL含有10μMOligo dT单链DNA,1X PNK反应缓冲液和66μMATP的反应中,并将[γ- 32 P] ATP反应在37°孵育30分钟将其置于冰上,并使用Sambrook和Russell的方法(Molecular Cloning,v3,2001,pp.A8.25-A8.26)进行分析。

 

蛋白质浓度(OD 280)测量使用使用2.0mg / ml BSA样品(Pierce Cat#23209)标准化的Nanodrop ND-1000分光光度计
在OD 280处分析2.0μL酶样品并用产品储存溶液冲切。使用消光系数63,440和分子量34,620道尔顿将观察到的3个重复样品的平均测量结果转换为mg / mL。

 

SDS-Page(物理纯度评估)
将2.0μL酶溶液加载到变性的4-20%Tris-甘氨酸SDS-PAGE凝胶上,其侧翼为宽范围MW标记和2.0μL1:100稀释的样品。电泳后,将凝胶染色并比较样品以确定物理纯度。该测试的验收标准要求浓缩样品中污染带的总量不超过稀释样品中目的蛋白质条带的质量,确认浓缩样品的纯度大于99%。

 

 

部分产品信息:

货号

品名

规格

浓度

品牌

N2070-10-L

ATP Solution 

50 µmol

10 mM

Enzymatics

N2050-10-L

10 mM dNTP Solution Mix

100 µmol

10 mM

Enzymatics

N2010L

100 mM 75µmol dNTP Solution Set 

75 µmol

100 mM

Enzymatics

N2010-A-L

100 mM dATP Solution 

75 µmol

100 mM

Enzymatics

N2050L

100µmol dNTP Solution Mix 

100 µmol

25 mM

Enzymatics

Y9180L

10x Uracil Cleavage System 

750 Reactions

N/A

Enzymatics

P7670

2x HiFi PCR Master Mix 

1 rxn

2x

Enzymatics

Y9420L

5x ER/A-Tailing Enzyme Mix 

24 reactions

N/A

Enzymatics

Y9380L

E. coli Pyrophosphatase 

50 U

100 U/mL

Enzymatics

Y9030L

E. coli Single-Stranded DNA Binding Protein 

1.5 mg

2.75 mg/ml

Enzymatics

Y9140-HC-L

End-Repair Mix (High Concentration) 

75 Reactions

1 Rxn/µL

Enzymatics

Y9140-LC-L

End-Repair Mix (Low Concentration) 

200 Reactions

1 Rxn/µL

Enzymatics

Y9080L

Endonuclease VIII 

10,000 U

10,000 U/ml

Enzymatics

P7600L

EnzScript™ (MMLV Reverse Transcriptase RNase H-) 

10,000 U

200,000 U/mL

Enzymatics

X8010L

Exonuclease I 

30,000 U

20,000 U/ml

Enzymatics

X8020L

Exonuclease III 

50,000 U

100,000 U/ml

Enzymatics

P7010-HC-L

Klenow (3′→ 5′ exo-) (High Concentration) 

10,000 U

50,000 U/ml

Enzymatics

P7010-LC-L

Klenow (3′→ 5′ exo-) (Low Concentration) 

10,000 U

5,000 U/ml

Enzymatics

P7060L

Klenow Fragment 

2,500 U

5,000 U/ml

Enzymatics

X8030L

Lambda Exonuclease 

10,000 U

5,000 U/ml

Enzymatics

P7040L

M-MuLV Reverse Transcriptase 

100,000 U

200,000 U/ml

Enzymatics

P7090L

Mako DNA Polymerase (3→ 5 exo-) 

3,000 U

30,000 U/ml

Enzymatics

P7140-HC-L

Manta 1.0 DNA Polymerase (High Concentration) 

100,000 U

400,000 U/ml

Enzymatics

P7140-LC-L

Manta 1.0 DNA Polymerase (Low Concentration) 

100,000 U

40,000 U/ml

Enzymatics

P7610L

NGS Library Prep: 2X VeraSeq™ PCR Mix – 250 Reactions 

250 Reactions

2X

Enzymatics

P7590L

Phoenix™ Hot Start Taq DNA Polymerase 

500 U

5,000 U/ml

Enzymatics

P7460L

Poly(A) Polymerase 

1,000 U

5,000 U/mL

Enzymatics

Y9260L

RecA 

1.5 mg

2.0 mg/mL

Enzymatics

Y9220L

RNAse H 

5,000 U

5,000 U/ml

Enzymatics

Y9240L

RNAse Inhibitor 

20,000 U

40,000 U/ml

Enzymatics

L6010L

T3 DNA Ligase 

900,000 U

3,000,000 U/ml

Enzymatics

L6030-LC-L

T4 DNA Ligase 

150,000 U

120,000 U/ml

Enzymatics

L6030-HC-L

T4 DNA Ligase (Rapid) 

240,000 U

600,000 U/ml

Enzymatics

P7080L

T4 DNA Polymerase 

2,000 U

3,000 U/ml

Enzymatics

Y9130L

T4 Gene 32 Protein 

1.0 mg

10 mg/mL

Enzymatics

Y9040L

T4 Polynucleotide Kinase 

10,000 U

10,000 U/ml

Enzymatics

L6050L

T4 RNA Ligase 1 

10,000 U

20,000 U/ml

Enzymatics

L6080L

T4 RNA Ligase 2 

4,500 U

30,000 U/ml

Enzymatics

L6070L

T4 RNA Ligase 2 Truncated 

500 U

5,000 U/ml

Enzymatics

L6020L

T7 DNA Ligase 

900,000 U

3,000,000 U/ml

Enzymatics

P7260L

T7 DNA Polymerase 

3,500 U

10,000 U/ml

Enzymatics

P7180L

T7 RNA Polymerase 

50,000 U

50,000 U/ml

Enzymatics

L6060L

Taq DNA Ligase 

20,000 U

40,000 U/ml

Enzymatics

P7110L

Taq DSC 2.0 DNA Polymerase 

1,250 U

5,000 U/ml

Enzymatics

P7250L

Taq-B DNA Polymerase 

10,000 U

5,000 U/ml

Enzymatics

P7620L

TaqIT 

5,000 U

50,000 U/mL

Enzymatics

P7070L

Terminal deoxynucleotidyl Transferase (TdT) 

6,000 U

20,000 U/ml

Enzymatics

G5020L

Thermolabile UDG 

500 U

1,000 U/ml

Enzymatics

Y9370L

Thermostable Pyrophosphatase 

1,250 U

2,000 U/ml

Enzymatics

G5010L

Uracil DNA Glycosylase 

10,000 U

2,000 U/ml

Enzymatics

P7511L

VeraSeq™ 2.0 High-Fidelity DNA Polymerase 

500 U

2,000 U/ml

Enzymatics

P7520L

VeraSeq™ ULtra DNA Polymerase 

500 U

2,000 U/ml

Enzymatics

L6030-W-L

WGS Ligase 

24 Reactions

N/A

Enzymatics

P7640L

ZipScript™ One-Step RT-qPCR Kit 

1000 Reactions

N/A

Enzymatics

P7020-HC-L

φ29 DNA Polymerase (High Concentration) 

2,000 U

100,000 U/ml

Enzymatics

P7020-LC-L

φ29 DNA Polymerase (Low Concentration) 

2,000 U

10,000 U/ml

Enzymatics

 

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