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蚂蚁淘在线 / 品牌中心 / Lucigen / Lucigen/Expresso® T7 Cloning and Expression System, C-His/49002-2/10 rxns

Lucigen/Expresso® T7 Cloning and Expression System, C-His/49002-2/10 rxns

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￥7640.00
货号：49002-2
浏览量：127
品牌：Lucigen
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商品描述
CloneandexpressevendifficultproteinswiththefastestandeasiestdirectionalPCR-basedcloningsystemavailable.
  • Savetime!Enzyme-freecloninginsecondswithcloning-readyvectorandcells.
  • Highefficiency:›90%recombinants.
  • Tightly-controlledexpressionofN-orC-terminal6xHistaggedproteins.

AlsoavailablewithcleavableSUMOSolubilityTag

CompleteCloningandExpressionSystem

TheExpressoT7CloningandProteinExpressionSystemsaredesignedforfast,easy,andefficientdirectionalcloningandexpressionofPCR-amplifiedgenes.Thesystemsarecompletewithpre-processedpETite™N-orC-Hiscloningvectors,andtwocompetentcelllines,suppliedinsingletransformationvials.HighefficiencyHI-Control™10GChemicallyCompetentCellsenablestablecloningandHI-ControlBL21(DE3)CompetentCellsprovidetightlycontrolledproteinexpression,thushelpingyouavoidexpressionproblemsseenwithleakyT7promoter-basedsystems.TheN-orC-terminal6xHistaggedproteinscanberapidlypurifiedusingstandardNichromatography.SystemsarealsoavailablethatencodeanN-terminal6xHisSUMOtagfusionforimprovedsolubilityofexpressedprotein.OtherExpressoSystemsareavailablewhichexpressproteinsunderthecontroloftheRhamnosepromoter.

Five-SecondDirectionalCloningofPCR-AmplifiedGenes

TheExpressoT7CloningandProteinExpressionSystemusesExpressioneeringTechnology™,anenzyme-freerecombinationalcloningstrategytoseamlesslyintegratethegenewiththevector.ThetargetgeneisamplifiedbyPCRusingprimersthatadd18base-pairsofvector-complementarysequencetobothendsofthegene.Unlikeotherrestrictionenzymebasedmethodsorligase-freecloningmethods,nofurthercleanuporenzymatictreatmentofthePCRproductisnecessary.Simplymix1µloftheunpurifiedPCRreactionwiththesuppliedpre-processedpETite™T7expressionvector,andtransformimmediatelyintotheHI-Control™10GChemicallyCompetentCellsprovided(Figure1). 

Expresso saves you a day!


Cloning-ReadyVectorsSaveHoursofResearchTime.

NewpETiteT7vectorsinclude:

  1. StrongT7promoterforhigh-levelexpression.
  2. ChoiceofN-terminalorC-terminal6xHisfusiontagsforfastproteinpurification.
  3. Smallsize(2.2kb)foreasierdownstreammanipulation.
  4. PatentedCloneSmart®technologyincreasescloningefficiency.

pETite vector maps

Figure2.pETiteT7expressionvectors:Smallsize(2.2kbvs.5.4kbforpET)foreasiermanipulation,includingtargetedmutagenesis.Vectorsarepre-processedforinstantenzyme-freecloningofPCRproductswithachoiceofamino-terminalorcarboxyl-terminalfusionof6xHistagtoproteinofinterest.

HighcloningefficiencywithHI-Control10GChemicallyCompetentCells.

ThehightransformationefficiencyofHI-Control10GChemicallyCompetentCellsensuresrecoveryofcloneswithprecisejunctionsandthecorrectorientation.Formostgenes,>90%ofcolonieswillhavethetargetgeneinsertedinthecorrectorientation.(Figure3).

High cloning efficiency with Expresso T7 System

Figure3.HighcloningefficiencywithExpressoT7System.
Pre-processedpETiteC-Hisvectorwasmixedwith1µlofunpurifiedPCRgeneproductandtransformedintoHI-Control10GChemicallyCompetentCells.ColonyPCRwasperformedon18randomlychosencolonies;17of18containedinsertofthecorrectsize.

HI-ControlBL21(DE3)CellsControlLeakyProteinExpression

HI-ControlBL21(DE3)CellscontainhighlevelsoflacrepressortomaintaintightcontroloverexpressionofT7RNApolymerase.Tightercontrolmeansbettertoleranceofpotentiallytoxicgeneproducts(Figure4).

Expresso vs. pET28a

Figure4.ExpressionofvariousproteinsusingtheExpressoT7SystemversusapETVector.GenesencodingaDNApolymerase,abluefluorescentprotein(BFP),orATPsynthasebsubunit(membraneprotein)wereclonedintopET28aorpETitevectorswithN-terminalorC-terminal6xHistags(asindicated).pET28aclonesweretransformedintostandardBL21(DE3)cells,andpETiteclonesweretransformedintoHI-ControlBL21(DE3)cellsforexpression.CulturesweregrowninLBat37°CtoanOD600of0.5to0.7(odd-numberedlanes)andinducedfor3hourswith1mMIPTG(even-numberedlanes).CellswerepelletedandlyseddirectlyinSDS-PAGEloADIngbuffer,and0.05ODequivalentswereanalyzedbygelelectrophoresis.ThegelwasstainedwithCoomassieblue.

PurificationofActiveSolubleFluorescentProteinUsing6xHisTag

TheN-orC-terminal6xHistaggedproteinsexpressedusingtheExpressoT7CloningandExpressionSystemcanberapidlyaffinity-purifiedovercommerciallyavailablenickelresins.Anexampleofthepurificationofa6xHistaggedyellowfluorescentproteinclonedandexpressedusingtheExpressoT7SystemisshowninFigure5. 

Expression and purification of active soluble fluorescent protein.

Figure5.Purificationofa6xHistaggedfluorescentprotein.HI-ControlBL21(DE3)cellsharboringpETiteC-Hisvectorcontainingayellowfluorescentprotein(YFP)geneweregrownat37°CinLBmediatoanOD600of0.6(lane1),theninducedwith
1mMIPTGfor4hours(lane2). Cellswereharvestedandlysedbysonicationin300mMNaCl,50mMTris-HClpH8.0.ClearedlysatewasloadedontoanNi-NTASepharose®Column.Columnflow-through(lane3,FT)andwash(lane4,W)fractionswerecollected.TheboundYFP,showninthecolumnatleft,waselutedwithwashbuffercontaining300mMimidazole(lanes5-12,E1-E8).Fluorescentfractionsalignwiththepureproteinasshownonthegel.

ImportantProductUseInformation:
ThisproductisthesubjectofU.S.Patent#6,709,861.AdditionalpatentapplicationsownedbyLucigenCorporationarepending.

The6xHistagislicensedfromHoffmann-LaRoche,Inc.,Nutley,NJand/orHoffmann-LaRocheLtd.,Basel,Switzerlandandisprovidedonlyfortheuseinresearch. InformationaboutlicensesforcommercialuseisavailablefromQiagenGmbH,QIAGENStrasse1,D-40724Hilden,Germany.Purificationofthe6xHistaggedproteinswithNi-NTAmanufacturedbyQIAGENishighlyrecommendedforbestperformancesandtoavoidpoorpurificationresults.

ORDERINFORMATION

TheExpressoT7Cloning&ExpressionSystemcontainspre-processedpETite®N-Hisand/orpETiteC-HisVectorDNA,HI-Control™10GChemicallyCompetentCellsforcloning,andHI-ControlBL21(DE3)ChemicallyCompetentCellsforproteinexpression.AlsoincludedareN-Hisand/orC-HisPositiveControlInsertDNAs,andtransformationpositivecontrolpUCDNA.

TheExpressoT7SUMOCloningandExpressionSystemcontainspre-processedpETite®N-HisSUMOVectorDNA,HI-Control™10GChemicallyCompetentCellsforcloning,andHIControlBL21(DE3)ChemicallyCompetentCellsforproteinexpression.AlsoincludedareSUMOPositiveControlCInsertDNA,transformationpositivecontrolpUCDNA,SUMOExpressProtease,SUMOCleavageControlProtein,andforwardandreversePCRprimerstoconfirmclones.

Lucigen基因组编辑和工程面临许多挑战。为您的编辑实验获取可靠的试剂不应该是其中之一。无论您是在突破CRISPR技术的界限，在体内产生基因敲除或敲入还是在进行序列消化或鉴定的体外反应，您都需要可靠的工具和酶。依靠CRISPRcraft™获得质量，可靠性和稳定的性能。