Coomassie777tk

anewsletterforgenecloning,expression,andanalysisvolume10issue4june20035GeneratepETconstructsinlesstime10Expressnativeortaggedproteinsfromonevector13PanVeraacquisi-tionincreasesyourresources19ImproveyourexpressionprofilingFreeyourhandsFreeyourhandsBaculoDirectBaculovirusExpressionSystemrequireslesshands-ontime18009556288Expressions10.
4newproduct2uniqueinspeedTraditionalbaculovirussystemsrequirebac-terialtransformationandisolationofalargebacmidorco-transfectionofbaculoviruslin-earDNAandatransfervectorintoinsectcells.
TheBaculoDirectSystemgivesyourapidresultsbyeliminatingthesetime-con-sumingsteps.
Frominitialtransfectiontoisolationofyourbaculovirusstocktakesonly8hoursofhands-ontime—lessthanhalfthetimerequiredwithtraditionalbac-ulovirussystems.
simplifiedcloningTheBaculoDirectSystemutilizesGatewayTechnologytostreamlineyourresearch.
ThespeciallyengineeredBaculoDirectLinearDNA(baculovirusgenome)containsattRsitesforefficientrecombinationwithaGatewayentryclone*containingyourgeneofinterest.
Justmixtheentryclonecontain-ingyourgeneofinterestwiththeBaculoDirectLinearDNAandLRClonaseEnzymeMixforonehouratroomtempera-ture.
Theresultingreactionmixcontainstherecombinantbaculoviruscarryingyourgeneofinterest.
Usethisreactionmixtodirectlytransfectinsectcells.
Afterseventy-twohoursofgrowth,you'llhaveyourrecombi-nantbaculovirusreadyforexpressionoramplification.
Baculovirusexpressionhasneverbeeneasier.
analyzemoreUnlikeotherbaculovirussystems,theBaculoDirectSystemistrulyamenabletohigh-throughputexpressionscreening.
GatewayentryclonesandtheBaculoDirectLinearDNAcanbearrayedinmultiplewells,enablingyoutoperformtheLRreaction,transfection,andinfectionofinsectcellsinmulti-wellplates.
Thisallowsyoutoanalyzemultipleproteinssimultaneously.
You'llsavetimeandgetyourresultsfaster.
parent-freevirusTheBaculoDirectLinearDNAincludestwobuilt-intime-savingchecks—thethymidinekinase(TK)geneandlacZgene—toensuretheisolationofapurebaculovirusstock(fig-ure1).
DuringtheLRreactionwithyourGatewayentryclone,theTKandlacZgenesarerecombinedoutasby-productsofthereaction.
Cellsarethenplacedundertheselectionofganciclovir,adrugthatselectsagainstviruscontainingtheTKgene.
Ganciclovirefficientlyeliminatesanyremainingparental,non-recombinantvirus.
Thepurityoftheviralstockcanbequicklydeterminedbystainingcellswithβ-galac-tosidasetorevealanyremaininglacZ-con-tainingparental,non-recombinantvirus.
WiththeBaculoDirectSystem,you'llhaveapurevirusstockinonlyoneweekandelimi-natetheneedformultipleroundsofplaquepurificationtoisolaterecombinantvirus.
hands-freeexpressionSavetimeandgettoyourexpressionresultsfasterwiththeBaculoDirectBaculovirusExpressionSystem.
Callandordertoday.
*TocreateaGatewayentryclone,cloneyourgeneofinterestintoaGatewayentryvectorusingPCRcloningortraditionalrestrictionenzymedigestandligation.
Formoreinformation,visitwww.
invitrogen.
com/gateway.
TheBaculoDirectExpressionKitincludesBaculoDirectLinearDNA,pENTR/CAT(Gatewayentrycloneexpressioncontrol),CellfectinReagent,LRClonaseenzymemix,frozenSf9insectcells,Grace'sMedium,andganciclovir.
TheBaculoDirectTransfectionKitincludesBaculoDirectLinearDNA,CellfectinReagentandpENTR/CAT.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
FreeyourhandswithsimplifiedbaculovirusexpressionheBaculoDirectBaculovirusExpressionSystemmakesbaculovirusexpressionfasterandeasierthaneverbefore—savingyouhoursoftimewhencomparedtootherbaculovirussystems.
WiththeBaculoDirectSystem,you'llgetrapidresultswithlesseffortandfewersteps.
TBaculoDirectBaculovirusExpressionSystemProductQuantityCat.
no.
BaculoDirectExpressionKit5reactions12562-013BaculoDirectTransfectionKit5reactions12562-039figure1-engineeredBaculoDirectLinearDNAforsimplified,fastbaculovirusexpressionBaculoDirectLinearDNAattR1TkgenelacZattR2V5-HisPIE-OPPIOPPHAocIExpressions10.
4www.
invitrogen.
comnewproduct3SuperScriptIndirectcDNALabelingSystemcDNAyieldaffectsmicroarrayresultscDNAlabelingmethodsreproducemRNApopulationsinisolatedsamples.
Toeffec-tivelymonitorsubtlechangesingeneexpression,itisimportanttomaximizethenumberofmRNAtranscriptsthatarereversetranscribedintocDNAduringthelabelingreaction.
Full-lengthcDNAsareoptimalbecausetheyallowyoutoanalyzeinternaland5regionsofgenesofinterest.
TheperformanceofthereversetranscriptaseisthemostintegralaspectaffectingcDNAyieldandlength.
TheSuperScriptIndirectSystemusesSuperScriptIIIRTtoconsis-tentlygenerateincreasedyields(figure1)andfull-lengthcDNA.
ThisensuresthatthelabeledcDNAaccuratelymatchesyourmRNAtranscriptpopulationandincreasesthesensitivityofyourexperiment.
signalintensityisimportantinmicroarraystudiesManygenesinvolvedincriticalcellularpath-waysareexpressedatlowtomediumabun-dancelevels.
Usingasensitivelabelingmethodthatprovidesstrongsignalintensityenablesthedetectionoflowabundancetran-scripts.
TheSuperScriptIndirectSystemusesauniqueamino-allyl/aminohexylmodifiednucleotide,mixtureduringcDNAsynthesisresultinginefficientandevenmoreincorpo-rationofamino-modifiednucleotidesforcou-pling.
LabeledcDNAtargetshybridizemoreefficientlyandexhibitstrongfluorescentsig-nals,increasingsensitivity(figure2).
don'twaitThereisnoreasontocompromiseonper-formance.
UsingtheSuperScriptIndirectcDNALabelingSystem,you'llobtaingreatercDNAyieldsandincreasedsignalintensity,improvingthesensitivityofyourmicroarrayexperiments.
CallInvitrogenandordertoday.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
IncreasemicroarraysensitivitywiththeSuperScriptIndirectcDNALabelingSystemheSuperScriptIndirectcDNALabelingSystemincreasesthesensitivityofyourmicroar-rayexperiments.
Basedonwidelyused,provenlabelingprotocols,thesystemincludesSuperScriptIIIRTandauniquenucleotidemixture,ensuringbetteryieldsandbrightsignals.
TProductQuantityCat.
no.
SuperScriptIndirectcDNALabelingSystem10rxnsL1014-0130rxnsL1014-02figure1-SuperScriptIndirectcDNALabelingSystemgenerateshighercDNAyieldsthanthecompetition10001500200025003000500First-StrandcDNASynthesis(pmol)0CompetitorASuperScriptIndirectcDNALabelingSystemCompetitorBFirst-strandcDNAwassynthesizedfrom10gHeLatotalRNA,usingtheSuperScriptIndirectcDNALabelingSystemandvariouscommercialkitsaccordingtomanufacturers'protocols.
Ineachreaction,32P-α-dCTPwasaddedtotracethecDNAsynthesisand20%ofthereactionmixturewasspottedonGF/Cfil-ters.
cDNAyieldwascalculatedaccord-ingtoTCA-precipitated32Pcounts.
figure2-SuperScriptIndirectcDNALabelingSystemgivesyoubrightersignalsthanthecompetitionMeansignal-to-noiseratio01015205SuperScriptIndirectcDNALabelingSystemCompetitorACompetitorBAnalysisofarrays(MWGHumanStarter)hybridizedwithCy3-labeledcDNApreparedusingtheSuperScriptIndirectcDNALabelingSystemandcompetitorkitsaccordingtomanufac-turers'protocols.
Triplicatelabelingreactionswereperformedandhybridizedtoarray.
Graphshowsthemeansignal-to-noiseratios.
18009556288Expressions10.
4productreview4VerveMammalianTwo-HybridKitwithTOPOToolsTechnologyRapidandaccuratemammaliantwo-hybridanalysischievetwo-hybridanalysisresultsinanativemammalianenvironmentfasterwiththeVerveMammalianTwo-HybridKitwithTOPOToolsTechnology.
Thisuniquemethodreplacestime-consumingtraditionalcloningstepswitha10-minutereaction.
You'llquicklygeneratebaitandpreyconstructs,savinguptofourdaysofresearchtime.
AabettersystemMammaliantwo-hybridsystemsenablethedetectionandvalidationofprotein-proteininteractionsinanativeenvironment.
Typically,thegeneencodingtheproteinofinterest("bait")isfusedtoaDNAbindingdomain.
Thegeneencodingthepotentialinteractingpartner("prey")isfusedtoanactivationdomain.
Theseconstructsarethenco-transfectedintoamammalianhostalongwithareporterconstruct.
Whenthe"bait"and"prey"interact,thereportergeneisexpressed.
Usingconventionalmammaliantwo-hybridmethods,you'llspenddayscloninggenestogeneratethebaitandpreyconstructs.
TheVerveMammalianTwo-HybridKitwithTOPOToolsTechnologyreplacesthesetime-consumingstepswithPCRanda10-minuteTOPOJoiningreac-tion.
You'llcreatelinear"bait"and"prey"constructsinonedayandhaveresultswith-in24-72hours.
afasterprocedureUsingTOPOToolsTechnology,therearenoligations,novectormanipulations,nocloning,andnoE.
colitransformations,sav-ingdaysoftime.
SimplyPCRamplifyyour"bait"and"prey"genes,jointhe"bait"producttothePSV40/GAL45andSV40pA3elementsandthe"prey"producttothePSV40/VP165andSV40pA3elementsina10-minuteTOPOJoiningreaction,andco-transfectthelinearconstructsandareporterplasmidintothemammalianhost(figure1).
accuratelydetectinteractionsTodemonstrateaccuratedetectionofproteininteractions,theVerveTwo-HybridKitandaplasmid-basedsystemwereusedtodetecttheknowninteractionbetweenp53andthelargeTantigeninCHOcells.
Thepositiveinteractionwasdetectedinbothsystems(figure2),butresultswereachieveddaysearlierusingtheVerveKit.
easyhigh-throughputSincetherearenotime-consumingcloningsteps,youcaneasilyadapttheVerveTwo-Hybridproceduretoahigh-throughputfor-matandanalyzehundredsofinteractions.
speedtotwo-hybridanalysisGetfasterinteractionresultswiththeVerveMammalianTwo-HybridKitwithTOPOToolsTechnology.
Ordertoday.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
ProductQuantityCat.
no.
VerveMammalianTwo-HybridKitwithTOPOToolsTechnology100rxnsT501-100figure2-detectionofthep53:largeTantigeninteraction10203040500p53p53TAgCPplasmidbait:prey:DNAform:p53p53TAgCPVervelinearconstructsFoldinductionofβ-galactivityThep53-baitwastestedwitheitherSV40TAg-prey,oranegative-controlCP-prey,asindicated.
Baitandpreyconstructswereco-transfectedusingLipofectamine2000Reagent.
β-galactosidaseactivitywasmeasured48hoursposttransfectioninaCHOcelllinecontainingthestablyinte-gratedpGAL/lacZreportergeneconstruct.
figure1-overviewoftheVerveMammalianTwo-HybridKitmethodreportergeneE1bTATAGAL4UASLacZZAmpicillinZeocinpUCoriSV40pASV40oriEM7E1bTATALacZGAL4UASpGAL/lacZBGHpA5DBDElementReporterplasmidBaitproteinexpressionPreyproteinexpressionSV40pA3Element5ADElementgeneofinterestSV40pA3ElementCreateanexpressioncassetteforthepreyandbaitprotein1Assayforreporterexpression3Cotransfectbothexpressioncassettesandthereporterplasmidintomammaliancells2geneofinterestExpressions10.
4www.
invitrogen.
comproductreview5ChampionpETExpressionSystemThefastestwaytogeneratehigh-levelpETexpressionconstructsaximumproteinyieldsfromtheChampionpETExpressionSystemmakeitthemostpowerfulsystemforexpressingrecombinantproteins.
NowcloningintoaChampionpETvectorisfasterandmoreefficientwiththeDirectionalTOPOCloningandGatewayTechnologies.
You'llgetyourclonequicklyandmoveontoexpressionsoonerthaneverbefore.
MVectorCat.
no.
PositionTagCleavageproteaseAntibioticresistanceAdvantagepET100/D-TOPOK100-01N-term6xHis-XpressEKAmpCleavabledetectionandpurificationtagpET101/D-TOPOK101-01C-termV5-6xHis–AmpDetectionandpurificationtagpET102/D-TOPOK102-01N-termThioredoxinEKAmpCleavablethioredoxintagenhancesproteintranslationandsolubilityC-termV5-6xHis––DetectionandpurificationtagpET151/D-TOPOK151-01N-termV5-6xHisTEVAmpCleavabledetectionandpurificationtagpET200/D-TOPOK200-01N-term6xHis-XpressEKKanCleavabledetectionandpurificationtagVectorsaresoldaspartofacompletekitthatincludeslinearized,topoisomerase-activatedvector,PCRreagents,OneShotTOP10E.
coli,andBL21Star(DE3)OneShotE.
coli.
table1-ChampionpETvectorsadaptedwithDirectionalTOPOCloningTechnologydomorewiththeChampionpETvectorsUsingtheChampionpETExpressionSystem,you'llrapidlyproducethehighestlevelsofprotein.
High-levelexpressionisaccomplishedviathestrongT7lacpromoterintheChampionpETvectorsandaspeciallydesignedBL21strain(BL21StarE.
coli,seebox).
Builtforspeed,thevectorsreplacetraditional,inefficientrestrictionenzymecloningmethodswithtwofast,innovativeoptions:DirectionalTOPOCloning—cloneyourgeneofinterest(goi)inthecorrectorien-tationforexpressioninfiveminuteswith>90%efficiencyGatewayTechnology—recombineyourgoiintoapETvectorinjustonehour;quicklytransferyourgeneintoanynumberofexpressionsystemswithoutsubcloningYou'llhaveyourexpressionconstructsoon-erandmoveontoexpressionrightaway.
fast,directionalcloningChampionpETDirectionalTOPOvectors(table1)providerapid,efficientcloningofPCRproductsusingtheligationfunctionoftopoisomeraseI.
SimplyaddyourPCRprod-uctdirectlytotheChampionpETDirectionalTOPOvector,incubatefor5minutes,andtransformintoE.
coli.
That'sit.
Greaterthan90%ofthePCRproductswillbeinthecorrect5→3orientation.
Thisspeedandefficiencymeansyou'llhaveyourexpressioncloneinjustoneday.
rapidrecombinationincreasesyouroptionsGatewayTechnology*enablesyoutoshuttleyourgoiintoasmanyvectorsandhostsys-temsasyouchoose—withouttime-consum-ingsubcloningsteps.
Recombinationisfastandefficient,takingjustonehourandmain-tainingdirectionality.
TheChampionpETGatewayvectorsincludeattrecombinationsitessothatyoucantakeadvantageofthisuniversalcloningandexpressionplatform.
fastcloningleadstospeedyresultsGenerateyourT7expressionclonefasterandmoreefficientlyusingtheChampionpETExpressionSystem.
You'llgetyourclonesoonerandmoveontohigh-levelexpressionearlierthaneverbefore.
Usetable1belowortheinformationonpage14toorderyourChampionpETvectortoday.
*FormoreinformationonGatewayTechnology,seethearticleonpages14-15orvisittheon-linetutorialatwww.
invitrogen.
com/gateway.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
TheBL21Star(DE3)OneShotChemicallyCompetentcellsincludedintheChampionpETExpressionSystemimproveyourexpres-sionyields.
BL21Star(DE3)E.
colimaxi-mizethepowerofT7transcriptionbypre-ventingdegradationoftranscribedmRNA.
ThisenhancedstabilitymeansmoremRNAisavailablefortranslation,resultinginincreasedproteinproduction.
!
BoostyourT7expressionyields18009556288Expressions10.
4applicationnote6SimplyBlueSafeStaintainingapolyacrylamidegelwithCoomassiebluestainisthemostcommonlyusedprocedureforvisual-izingproteinbands.
TraditionalCoomassiestainsareslowanddonotofferthesensitivitythatmanyexperimentsrequire.
SimplyBlueSafeStainisauniquelyformulated,ready-to-use,colloidalCoomassieG-250stainthatisspecificallydesignedtogivefast,sensitivestainingresults.
HerewedemonstratethatSimplyBlueyieldsresultsfive-timesfasterwithatleasttwicethesensitivityofothercommonlyusedCoomassiestainingreagents.
methodsGelelectrophoresis.
ThreeNuPAGENovex4-12%Bis-TrisGelswereidenticallyloadedwithdifferentamountsofvariousproteinsamples.
TheproteinswereseparatedusingMESSDSRunningBufferinanXCellSureLockMini-Cell.
Staining.
Afterelectrophoresis,twoofthethreegelswerewashedtwotimes,fivemin-uteseach,with100mlofultrapurewater.
Oneofthesegelswasstainedwith20mlofSimplyBlueSafeStain.
Theotherwasstainedwith20mlofGelCodeBlue(PierceChemicalCompany).
Bothgelswerestainedforonehour.
Thestainswerethendecantedandthegelsweredestainedin100mlofultrapurewaterforonehour.
Afteronehour,20mlofa20%NaClsolutionwasaddedtothewaterofthegelstainedwithSimplyBlueSafeStain.
Thethirdgelwasstainedwith20mloftheconventionalCoomassieR-250stain(0.
1%BrilliantblueR-250in50%ethanol,10%aceticacid)for30minutes.
Thisgelwasthendestainedin10%aceticacidfor30minutes.
Banddevel-opment,background,andsensitivityforeachgelwasanalyzedwithscannedimagestakenatvarioustimepointsintheprotocol.
resultsStainingtime.
TheresultsdemonstratethatwithSimplyBlueSafeStain,proteinsstainfive-timesfasterthanwithotherCoomassiestains(table1andfigure1,page7).
After5minutesintheSimplyBlueSafeStain,1gofproteinisdetected(GelA,lanes2and3).
After5minutesintheGelCodeBluestain,6gofproteinisonlyfaintlydetected(GelB,lane1).
UsingGelCodeBlue,the1gbandsareonlyvisibleafter25minutesinthestain(imagenotshown).
Although1gofproteincanbedetectedafter5minutesinthecon-ventionalCoomassieR-250stain(GelC,lanes2and3),backgroundishigh,obscur-ingthedetectionoflowerconcentrationsofproteinandtheMark12UnstainedStandard(GelC,lanes9and10).
Sensitivity.
SimplyBlueSafeStainisatleasttwotimesmoresensitivethanotherCoomassiestains(table1,figure1,page7).
Aftertwohoursinthewater-baseddestain,7ngofreducedBSAcanbedetectedonthegelstainedwithSimplyBlueSafeStain(GelD,lane7).
Only20ngofreducedBSAcanbedetectedonthegelsstainedwitheitherGelCodeBlueorconventionalR-250stain.
Whenleftovernightinthewater-baseddestain,aslittleas3ngofreducedBSAcanbedetectedonthegelstainedwithSimplyBlueSafeStain(GelG,lane8).
WhentheGelCodeBlue-stainedgelisleftovernightinwaterdestain,only20ngcanbedetected(GelH,lane5).
SlightlyincreasedsensitivityisseenonthegelstainedwithconventionalR-250stain(GelI,lane6).
continuedonpage7SimplyBlueSafeStainoutperformsthecompetitioninproteinstainingSAllenBautista,TechnicalServices,InvitrogenCorporation,Carlsbad,CASensitivitySimplyBlueGelCodeCoomassieSafeStainBlueR-250Comments5minutesinstain1gBSA6gBSA1gBSAMark12andlowconcentrationsofBSAweredetectedwithSimplyBlueSafeStain.
HighbackgroundwithR-250stain.
2hoursinrecommended7ngBSA20ngBSA20ngBSASimplyBlueSafeStaindemonstratesthehighestsensitivity.
destainingsolution*Overnightinrecommended3ngBSA20ngBSA>10ngBSANolossinsensitivitywhengelsarekeptovernightindestainingsolution*SimplyBluewater-baseddestain.
table1-speedandsensitivityresultswithdifferentCoomassiestains*DestainforSimplyBlueconsistsof20%NaClinwater.
DestainforGelCodeBlueconsistsofwaterasrecommendedbymanufacturer.
DestainforR-250Coomassiestainconsistsof10%aceticacidinwaterSpeedExpressions10.
4www.
invitrogen.
com7discussionSimplyBlueSafeStainprovidesfaster,moresensitivestainingresultsthanotherCoomassiestains.
Itallowsidentificationofproteinbandsquicklyandwithoutambigui-ty,enablingfurtheranalysistoproceedpromptly.
Achievethemaximum-perform-anceproteinstainingresultsyourexperi-mentsrequire.
CallInvitrogenandorderSimplyBlueSafeStaintoday.
*Sufficientreagentissuppliedtostain50mini-gels.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
SimplyBlueSafeStaincontinuedfrompage6figure1-comparisonofspeedandstainingsensitivityofthreeCoomassiestainsSimplyBlueSafeStainGelCodeBlueCoomassieR-250Stain123456789101234567891012345678910123456789101234567891012345678910123456789101234567891012345678910ABCDEFGHIThefollowingsampleswereelectrophoresedonNuPAGENovex4-12%Bis-TrisGelsandthenstainedwithSimplyBlueSafeStain,GelCodeBlue,orCoomassieR-250followingmanufacturer-recommend-edprotocols.
Itemstonotearelistedbeloweachgel.
Arrowsindicateachievedsensitivity.
Water-baseddestainSensitivity:≤7ngreducedBSANolossinsensitivitywithovernightwater-baseddestainingWaterdestainSensitivity:20ngreducedBSALossinsensitivitywithovernightwaterdestainingMethanol/aceticaciddestainSensitivity:>10ngreducedBSASensitivity:1gproteinMark12clearlyvisibleSensitivity:6gproteinMark12notvisibleSensitivity:1gproteinMark12notvisibleHighbackgroundWater-baseddestainSensitivity:7ngreducedBSAWaterdestainSensitivity:20ngreducedBSAMethanol/aceticaciddestainSensitivity:20ngreducedBSAHighBackgroundAfter5minutesinstainAfter2hoursindestainAfterovernightindestainLane1:6gproteinmixLane2:1grabbitIgGLane3:1greducedBSALane4:5gE.
colilysateLane5:20ngreducedBSALane6:10ngreducedBSALane7:7ngreducedBSALane8:3ngreducedBSALane9:10lMark12StandardLane10:5lMark12StandardProductQuantityCat.
no.
SimplyBlueSafeStain1L*LC60603.
5LLC6065Achieveultra-fastCoomassiestainingresultswiththeSimplyBlueSafeStainmicrowaveprocedure.
Detectaslittleas20ngin12minutes.
Seetheprotocolpull-outcardinthisissuefordetails.
20ngreducedBSAResultsin12minuteswiththeSimplyBlueSafeStainmicrowaveprocedure!
newcloningstrainsUntilnow,competentcellsfailedtoaddresstwocloningbottlenecks:slow-growingbacte-riaandhigh-efficiencytransformationsinaconvenientformat.
Notanymore.
Mach1-T1Rcellsgrowrapidly,allowingyoutoplateandpickcoloniesthesamedayortoperformmini-prepsfromafour-hourculture.
OmniMAX-T1RE.
coliisanimprovedDH5αstrainthatcombinesversatilegeneticmark-erswithhightransformationefficiency,enablingyoutogetyourclonefirsttime.
fastestgrowingstrainThinkofhowmuchfasteryoucouldclonegenesifyoudidn'twaitonovernightbacter-ialculturesorcouldplateandpickbacterialcoloniesthesameday.
Withthefastest-growingcloningstrain,Mach1-T1RE.
coli,thewaitisover.
Sufficientgrowthformini-prepsisachievedfromanovernightcolonyinjustfourhourscomparedtoovernightformostothercloningstrains,savingyouanentireday(figure1).
mostcompetentstrainOmniMAXT1Phage-resistantE.
colipro-videtransformationefficiencies>5x109cfu/g,givingyouabetterchanceofgettingyourdesiredclone(figure2).
Inaddition,thesecellslackmethylationrestrictionsys-tems,whichmeansyoucantransformbothmethylatedandnon-methylatedDNA.
BeingresistanttobothT1andT5phagesmeansyourculturesaresafefromphagecontami-nationandyourvaluablesamplesarepro-tected.
UseOmniMAX-T1Rcellsforunsur-passedresultsinyourcloningexperiments.
givethemashotMach1–T1RE.
colioffersyousignificantsavingsintimeduetoitsfastgrowthrate.
OmniMAX-T1RE.
coliisanimprovedDH5αstrainthatcombinesalltheimpor-tantgeneticelementsforcloningwiththehighesttransformationefficienciesavailable.
Usethesenewcompetentcellstotransformthewayyouclonetoday.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
18009556288Expressions10.
4newproduct8Mach1-T1RandOmniMAXT1RE.
coliClonegenesfasterandmoreefficientlywithtwonewcompetentcellsforcloningwonewE.
colistrainsincreasethespeedandimprovetheefficiencyofcloningnewgenes.
Mach1T1Phage-Resistant(T1R)chemicallycompetentE.
coliisthefastestgrow-ingcloningstrainonthemarket,savingyouvaluableresearchtime.
OmniMAXT1Phage-Resistant(T1R)chemicallycompetentE.
coliprovidethehighesttransformationefficiencies.
Withthesetwostrains,you'llclonegenesfasterandmoreefficientlythaneverbefore.
Tfigure1-timesavedusingMach1-T1RE.
coliTime(hrs)081216204Mach1-T1R*OthercloningstrainsTimetomini-prepTimetocolonyformationProductEfficiencyQuantityCat.
no.
OneShotMach1-T1RChemicallyCompetentE.
coli>1x10920x50lC8620-03MultiShotStripWellMach1-T1RChemicallyCompetentE.
coli>1x1091plateC8696-01OneShotOmniMAX-T1RChemicallyCompetentE.
coli>5x10920x50lC8520-03MultiShotStripWellOmniMAX-T1RChemicallyCompetentE.
coli>1x1091plateC8596-01figure2-OmniMAX-T1RcellsprovidesuperiortransformationefficiencyTransformationefficiencyofOneShotOmniMAX-T1R,XL10-Gold,andMAXEfficiencyDH5αcellswasdeterminedusing10pgpUC19plasmid.
Transformationswereperformedaccordingtomanufacturer'sproto-cols.
Valuesrepresentedaremean±SDoftriplicateobservations.
0123456798OmniMAX-T1RCompetentCellsTransformantsx109/gpUC19DNAXL10-GoldMAXEfficiencyDH5α*Withampicillinselection.
Expressions10.
4www.
invitrogen.
comnewproduct9SuperScriptIIIOne-StepRT-PCRSystemsforEndPointandReal-timeAnalysisprovenenzymesforgreatresultsBoththeSuperScriptIIIOne-StepRT-PCRSystemandtheSuperScriptIIIPlatinumOne-StepqRT-PCRKitcombineSuperScriptIIIRTandPlatinumTaqtogiveyouexcep-tionallysensitiveandspecificone-stepRT-PCR.
SuperScriptIIIRTisanRNaseH-mutantofSuperScriptIIRTthatexhibitsalongerhalf-life(220min.
at50°C)andincreasedthermostability,givingyouhighercDNAyields,greatersuccesswithRNAsec-ondarystructure,andincreasedspecificitywithgene-specificprimers.
PlatinumTaqDNAPolymeraseprovideshot-starttechnolo-gytoreducemisprimingforgreaterspecificityandsensitivityinyourPCR.
Whetheryou'redoingendpointorreal-timedetection,theseprovenenzymesguaranteegreatperformance.
sensitiveendpointanalysisTheSuperScriptIIIOne-StepRT-PCRSystemwithPlatinumTaqisoptimizedforsensitiveendpointanalysis.
The2Xreactionbufferandenzymemixhavebeenimprovedtoprovideincreasedsensitivity—downto0.
01pgtotalRNA,themostsensitivesystemavailable(figure1).
optimizedreal-timedetectionTheSuperScriptIIIPlatinumOne-StepqRT-PCRKithasbeendesignedandoptimizedtodeliverthebestreal-timeRT-PCRresults.
Aspeciallyformulatedbuffersystemproducessuperiorperformanceacrossmultipleinstru-mentplatforms,andprovidesyouwiththeflexibilitytodetectwithLUXFluorogenicPrimers*(figure2)ordual-labeledprobeswithoutcomprisingresults.
Inaddition,larg-erkitsizesprovidemorereactionstoaccom-modatehigh-throughputneeds,streamliningyourreal-timeexperiments.
takingone-stepfartherTakeyourone-stepRT-PCRfartherwithyourchoiceofhigh-performancekitsspecif-icallyoptimizedforendpointorreal-timeanalysis.
CallInvitrogenandordertheSuperScriptIIIOne-StepRT-PCRSystemortheSuperScriptIIIPlatinumOne-StepqRT-PCRKittoday.
*FormoreinformationonLUXFluorogenicPrimers,pleasevisitwww.
invitrogen.
com/lux.
TheseproductsmaybesubjecttooneormoreLimitedUseLabelLicenses.
PleaserefertotheInvitrogencatalogorwebsiteforthecorrespondingLimitedUseLabelLicenses.
Allproductsareforresearchuseonly.
Caution:Notintendedforhumanoranimaldiagnosticortherapeuticuses.
High-performanceone-stepRT-PCRforendpointandreal-timeapplicationsetthesuperioryieldofSuperScriptIIIReverseTranscriptaseandthehot-startspecificityofPlatinumTaqDNAPolymeraseintwonewone-stepRT-PCRkits.
ChoosetheSuperScriptIIIOne-StepRT-PCRSystemwithPlatinumTaqforsensitiveendpointanalysisortheSuperScriptIIIPlatinumOne-StepqRT-PCRKitforhigh-performancereal-timedetection.
GProductQuantityCat.
no.
SuperScriptIIIOne-StepRT-PCRSystemwithPlatinumTaq25rxns12574-018100rxns12574-026SuperScriptIIIPlatinumOne-StepqRT-PCRKit100rxns11732-020500rxns11732-088figure2-theSuperScriptIIIPlatinumOne-StepqRT-PCRKitisoptimizedforreal-timedetectionThehumanβ-actintranscriptwasquanti-fiedbyone-stepquantitativeRT-PCRfrom10-foldserialdilutionsofhumanHeLaRNA(100ngto0.
1pg)using200nMJOE-labeledLUXPrimer,200nMunlabeledprimer,SuperScriptIIIPlatinumOne-StepqRT-PCRkit,andROXReferenceDye.
Theresultingstandardcurveequationwas:y=-3.
410x+44.
166.
R2=0.
998.
0.
010.
101.
0005101520253035404550RNfigure1-theSuperScriptIIIOne-StepRT-PCRSystemwithPlatinumTaqpro-videssuperiorsensitivity0.
01GAPDH532bp0.
11MOne-StepRT-PCRreactionscontaining0.
01,0.
1,and1pgoftotalHeLaRNAwereperformedusingtheSuperScriptIIIOne-StepRT-PCRSystemwithPlatinumTaqDNAPolymerase.
RTincubationwas55°Cfor30minutes,followedby40cyclesofPCR,1min/kb.
18009556288Expressions10.
4newproduct10Tag-On-DemandTechnologytagitordon't,asyoupleaseDetectionofexpressedproteinsisoftenfacilitatedbytheuseofepitopetags.
Thisisparticularlyhelpfulwhenaprotein-specificantibodyforyourrecombinantproteinisnotavailable.
However,forsomeexperimen-talapproachesitisimportanttoexpressnativeprotein.
Inthepast,expressingyourrecombinantproteininbothepitope-taggedandnativeformsrequiredsubcloningyourgenetwiceandmaintainingtwovectorstocks.
Withthestate-of-the-artTag-On-DemandTechnology,youonlyhavetocloneyourgeneoncetoeasilyexpressyourrecombinantproteinwithorwithoutatag.
You'llgetnativeproteinexpresseduntilyouaddtheTag-On-DemandSuppressorSupernatant.
Thenyou'llproducehighlev-elsofyourproteintaggedwithGFPorV5.
It'seasytochoosehowyouwantyourpro-teinexpressedwithTag-On-DemandTechnology.
You'llsavetimeandefforttoo,byavoidingthoseextrasubcloningsteps.
high-performancevectorsTherearecurrentlytwomammalianTag-On-Demandexpressionvectorsavailable,pcDNA6.
2/V5-DESTandpcDNA6.
2/GFP-DEST(figure1),thatallowyoutotakeadvantageoftheTag-On-DemandTechnology.
Eachvectoroffers:AC-terminalV5orGFPepitopetagforefficientproteindetection—whenaddedtoyourproteiniftheopenreadingframe(ORF)usesanamber(TAG)stopcodonthatcanbesuppressedattsitesforuseinGatewayTechnologytoeasilytransferyourgeneintootherhostsystemsorvectorsCMVpromoterforhigh-levelexpressionInaddition,eachvectorincludestheBlasticidinresistancegeneforrapidandefficientselectionofstablecelllines.
ChoosetheV5antibodyepitopetagforextremelysensitivedetectionbywesternblottingortheGFPtagforeasyvisualdetectioninlivecells.
howitworksThekeytotheTag-On-DemandsystemistheSuppressorSupernatant.
Thisrecombi-nantadenoviralsupernatantdeliversasup-pressortRNAgenethatrecognizestheTAG,oramber,stopcodonpresentattheendofyourORF.
SimplyaddtheTag-On-DemandSupernatantdirectlytocellsthatcontainaTag-On-Demandexpressionvectortopro-ducetaggedprotein(figures2and3,page11).
IntheabsenceoftheTag-On-DemandSupernatant,theTAGstopcodonisrecog-nizedandyou'llexpressnativeprotein.
Nowyoucaneasilydetectyourproteinviaepitopeantibodyandconductsubcellularlocalizationstudiesviaimmunostainingorfluorescencewhenyouwant,orproducefullynativepro-teinfromthesameexpressionvector.
efficientsuppressionUpontheadditionoftheTag-On-DemandSuppressorSupernatant,you'llgetefficientandspecificsuppressionofTAGstopcodons,resultingingreaterthan50%ofyourrecombinantproteintaggedwithyourchoiceofV5orGFP(figure4,page11).
UsingaTag-On-DemandDESTvector,you'llgethighlevelsofproteinexpressionandefficienttaggingwhenyouwant,andnativeproteinwhenyoudon't.
ultimateconvenienceandcompatibilityTheTag-On-Demandvectorkitsareconve-nientlypackagedwitheithertheFlexibleproteinexpression—getnativeortaggedproteinondemandroducingbothnativeandepitope-taggedproteinfordifferentapplicationscurrentlyrequiressubcloningyourgeneintotwodifferentvectors.
Nowyoucaneliminateaddition-alsubcloningstepswithTag-On-DemandTechnology.
Expressnativeortaggedrecombinantproteinfromonevector,soyoucaneasilyandquicklygettoyourdownstreamstudies.
Pcontinuedonpage11figure1-Tag-On-DemandDESTvectorsattR2ccdBCmRattR1GFPT7pcDNA6.
2/GFP-DEST8kbpUCoriSV40pAPCMVAmpicillinTKpAEM7SV40orif1oriBlasticidinattR2ccdBCmRattR1V5epitopeT7pcDNA6.
2/V5-DEST7.
3kbPCMVAmpicillinpUCoriSV40pATKpAEM7SV40orif1oriBlasticidinExpressions10.
4www.
invitrogen.
com11pcDNA6.
2/V5-DESTorpcDNA6.
2/GFP-DESTvectorandTag-On-DemandSuppressorSupernatant.
UseanyGatewayentryclonethathasyourproteinexpressedwithaTAGstop.
Tag-On-DemandTechnologyisalsofullycompatiblewithanyoftheready-to-useUltimateHumanORFClonesprovidedassequence-verifiedGatewayentryclones.
SimplyrecombineyourgeneofinterestintotheTag-On-DemandDESTvectorofyourchoiceandyou'rereadytogo.
Formoreinfor-mationonUltimateORFClones,visitwww.
invitrogen.
com/clones.
demandthebestSaveyourtimetodotheexperimentsyouneed,nottocompleteextrasubcloningsteps.
WiththespeedandflexibilityofTag-On-DemandTechnology,you'llgetthenativeorepitope-taggedexpressionresultsyouneed.
Callandordertoday.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
Tag-On-DemandTechnologycontinuedfrompage10ProductQuantityCat.
no.
Tag-On-DemandGFPGatewayVectorKit1kitK410-01Tag-On-DemandV5GatewayVectorKit1kitK420-01Tag-On-DemandSuppressorSupernatant1kitK400-01figure2-howtheTag-On-DemandSuppressorSupernatantworksCMVGOI_+AddTag-OnDemandSuppressorSupernatantTransfectpcDNA6.
2/GFP-DESTorpcDNA6.
2/N5-DESTvectorcontaininggeneofinterestTAGstopNativeproteinTAGorV5GFPnon-TAGstopTAAorTGAGFPTaggedproteinAddasmallaliquotofTag-On-DemandSuppressorSupernatantatthetimeoftransfectionforexpressionofepitope-taggedprotein.
Yourgeneofinterest(GOI)withaTAGstopcodonshouldberecombinedintoapcDNA6.
2/V5-DESTorpcDNA6.
2/GFP-DESTvectorfromanyGatewayentrycloneoranyUltimateORFClone.
TheGOIwillbeplacedunderthecontroloftheCMVpromoterandin-framewithadown-streamV5orGFPepitopethatwillbefusedtotheC-terminusoftheprotein.
Theepitopetagisfollowedbyallthreestopcodons.
IntheabsenceoftheTag-On-DemandSuppressorSupernatant,nativeproteinisexpressedfollowingtransienttransfectionwiththeexpressionvector.
IftheTag-On-DemandSupernatantisadded,mostoftheproteinwillexpresstheepitopetag48hourspost-transfectionduetoreadthroughoftheTAGstopcodonandplacementofaserineresidueatthiscodon.
figure3-additionofaV5orGFPtagusingTag-On-DemandSuppressorSupernatantA:CHOcellsweretransfectedwithpcDNA3.
2/V5-GW/CATTAG,whichhasthechloramphenicolacetyltransferase(CAT)geneclonedintothepcDNA3.
2/V5-DESTvectorcompatiblewithTag-On-DemandTechnology,usingLipofectamine2000Reagent.
Proteinwasexpressedintheabsence(-)orpresence(+)oftheTag-On-DemandSuppressorSupernatant.
Forty-eighthourspost-transfection,20gofcelllysatewasanalyzedusinganAnti-V5antibodyonawesternblot.
B:Todemonstratethespecificityoftheadditionofthetag,293FTcellsweretransfectedwiththepcDNA6.
2/GFP-GW/CATTAGconstruct,whichhasthechloramphenicolacetyltransferase(CAT)geneclonedintothepcDNA6.
2/GFP-DESTTag-On-Demandvector,usingLipofectamine2000Reagent.
ProteinwasexpressedinthepresenceoftheTag-On-DemandSuppressorSupernatant(TAG)orsupernatantsexpressingthetRNAsuppressorsfortheTAAandTGAstops.
Forty-eighthoursposttransfection,20gofcelllysatewasanalyzedusinganAnti-GFPantibodyonawesternblot.
–+CAT-V5Anti-V5blotTAATAGTGACAT-GFPAnti-CATblotCATCATABfigure4-efficientepitopetaggingwithTag-On-DemandSuppressorSupernatant–+CAT-V5Anti-CATblotCATCHOcellswereco-transfectedwithapcDNA3.
2/V5-GW/CATTAGconstructusingLipofectamine2000Reagent,andproteinwasexpressedintheabsence(-)orpres-ence(+)oftheTag-On-DemandSuppressorSupernatant.
Forty-eighthoursposttransfec-tion,20gofcelllysatewasanalyzedusinganAnti-CATantibodyonawesternblot.
ThisshowsthatinthepresenceoftheTag-On-DemandSuppressorSupernatant,mostoftheexpressedproteinhasbeenshiftedupduetothepresenceoftheV5tag.
18009556288Expressions10.
4newproduct12UltimateMouseORFClonestime-savingmouseresourceTheUltimateMouseORFCloneCollectionconsistsofready-to-useclonesbuiltspecifi-callyforfunctionalanalysis.
EachUltimateORFCloneincludesonlythegenesequence,withoutaspecificpromoter,inthepENTR221Gatewayentryvectorforunlimiteddownstreamanalysisoptions.
Stringentqualitycontrolproceduresensurethecloneidentityandsequencevalidity.
There'snoneedforyoutospendyourtimeisolatingRNA,synthesizingcDNA,cloning,andverifyingsequence,savingweeksoftimeandeffort.
Justpurchaseyourclone(s)ofinterestandstartyourdownstreamstud-iesimmediately.
easilyenteranysystemWithGatewayTechnology,youcantransferyourgeneofinterestintodifferentsystemsandhostsforanalysis.
Asimpleone-hourrecombinationreactionmaintainsdirection-alityandsequencefidelity,eliminatingtime-consumingsubcloningandrevalidationsteps.
TheUltimateMouseORFClonesareprovidedinthepENTR221Gatewayentryvector(figure1),allowingyoutotakeadvantageofthisstate-of-the-arttechnology.
FromthisvectoryoucantransferyourgeneofinterestintoacomprehensiveselectionofGatewaydestinationvectorsforexpressionincell-free,E.
coli,yeast,insect,ormam-maliansystems.
totagornottotagInadditiontochoosingyourhost,theUltimateORFClonesallowyoutoexpressyourgenefromasinglevectorwithorwith-outaC-terminaltag.
*Eachgenecodingsequenceisfollowedbyanamberstopcodon.
Duringtranslation,thisstopcodonisrecognizedandyou'llexpressnativepro-tein.
AddingthespeciallyformulatedTag-On-DemandSuppressorSupernatanttotheexpressionculturesuppressesthisstopcodonandproducesaproteinfusedtothedownstreamvectorelements*.
Fromasinglevector,you'llbeabletoexpressnativeortaggedproteinsoyoucangettheresultsyouneed.
stringentrequirementsBeforeyoureceiveanUltimateMouseORFClone,itundergoesaseriesofqualitycon-troltests.
Allclonesarefull-insertsequencedandtheaminoacidsequenceisguaranteedtomatchthecorrespondingsequenceinGenBank.
Additionally,cloneidentityisconfirmedbeforeshipment.
Youcanbeconfidentthatthecloneyourequestisthecloneyoureceive.
findyourcloneon-linetodayManyUltimateMouseORFClonesarecur-rentlyavailable.
Tofindyourclone,visitwww.
invitrogen.
com/clones.
Injustafewclicksyoucanorderthecloneyouneedtodayandstartyourfunctionalanalysisstudiesquickerthaneverbefore.
*AppliesonlywhenrecombinedintoaTaq-On-Demandvector.
TolearnmoreaboutTag-On-DemandTechnology,checkoutthearticleonpage10ofthisissueofExpressions.
**Ordercloneson-lineatwww.
invitrogen.
com/clones.
Speedupyourresearchwiththeready-to-goUltimateMouseORFClonesheUltimateORFCloneCollectionconsistsoffull-length,full-insertsequencedopenreadingframes(ORFs)inaGatewayentryvector.
ThiscollectionnowincludesmouseORFs.
UsingtheUltimateMouseORFClones,youcanquicklybeginstudyingtheeffectsofyourgeneofinterestwithouttediouspreparationstepsandlimitingsingle-systemvectors.
TProductCat.
no.
UltimateMouseORFClonesMORF01figure1-UltimateORFcloneinthepENTR221GatewayentryvectorattL2KanamycinpUCoriattL1T2T1ORFpENTR221Expressions10.
4www.
invitrogen.
com13announcementproductreviewEvoQuestCustomPeptidesandAntibodiesInvitrogenacquiresPanVeraEasyon-lineorderingofcustomantibodiesandpeptidesowit'seasierthanevertoorderhigh-qualityEvoQuestcustompeptidesandantibodies.
Threenewon-lineorderingforms—forcustompeptidesynthesis,custompeptide-antibodyproduction,andantibodyproductionwithuser-suppliedimmunogen—taketheguessworkoutofyourproject.
atyourfingertipsOrderingpeptidesandantibodiestakesextensiveplanningandconsideration.
WhatscaleandpuritydoIuseformypeptideWhichconjugationstrategyisthebesttouseWhatanimalspeciesdoIusetoraiseantibodiesTogetthemostoutofyourproj-ect,youdon'twanttomissanyaspect.
Thenewon-lineorderingformsfromEvoQuestCustomServiceswalkyouthroughtheorderingprocess,soyoucanbeconfidentthatallyouroptionsarepresented.
orderingmadesimpleUsingtheon-lineorderingformsisstraight-forward.
Youcanaccessthetwoantibodyorderingforms(user-suppliedimmunogenorpeptidesynthesisandantibodyproduction)onthecustomantibodywebsiteatwww.
invitrogen.
com/polyclonal.
Thepeptideformislocatedatwww.
invitrogen.
com/cus-toms.
Allyouneedtodoistoregister,andausernameandpasswordwillbegiventoyou.
Theformswillthenguideyouthroughtheorderingprocessstepbystep(figure1).
nomoreguessingEachcustomon-lineorderingformtakesyouthroughthenumerousoptionsforpep-tideandantibodyproduction.
Easy-to-usedropdownmenusprovidelistsofavailablechoices(figure1).
You'llalsofindapricesheetwithabriefdescriptionofservices,instructionsonhowtoenterthepeptidesequencewithmodifications,anddirectionsfororderingpeptidedesign.
tryitoutThethreeon-lineorderingformsforcustomantibodyandpeptideservicestaketheguessworkoutofordering.
Trythemtodayatwww.
invitrogen.
com/polyclonalorcallEvoQuestcustomerserviceat8009556288ext.
45682.
hepowerfulnewpartnershipofInvitrogenandPanVeragivesyouadependablesingleresourceforabroadspectrumofproductlinestohelpyouattainyourdrugdiscoveryresearchgoalsmoresimply,moreeffectively,andmoreefficiently.
high-quality,dependablebiochemi-calassaysandpurifiedproteinsYoucanidentifypotentialtargetsmorerap-idlyandpreciselywithPanVera'srobustKinase,NuclearReceptor,andDrugMetabolismassaysandpurifiedproteins.
Thenewestoftheseproductsincludethe:reliable,easy-to-useZ-LYTEKinaseandPhosphataseAssayssensitive,reliableTerbiumChelateLabelingProductsfordetectingintermole-cularinteractionsdependable,sensitiveVividP450ScreeningKitsfordrugmetabolismresearchsensitivecell-basedassaysTwohighlyaccuratetechnologiesareavail-abletomeetyourcell-basedassayneeds:Beta-LactamaseAssays(BLA)formeasur-inggeneexpressioninmammaliancellsVoltageSensorProbes(VSPs)forionchannelresearchtorapidlydetectanytar-getthatchangesthemembranepotentialofacellasynergisticnewrelationshipYounowhavethecombinedresourcesoftwocompaniestohelpfurtheryourresearch.
Visitourwebsites(www.
panvera.
comorwww.
invitrogen.
com)formoreinformation.
TNfigure1-easy-to-useon-linecustomantibodyorderingformInvitrogenacquiresPanVera–moreresources,moreinnovation18009556288Expressions10.
4productreview14GatewayTechnologyComprehensiveselectionofGatewayexpres-sionvectorstomeetyourresearchneedsatewayTechnologyallowsyoutocloneyourgeneofinterestintoanentryvectorandthenshuttleit—withouttraditionalsubcloning—intoavarietyofexpressionvectors,sav-ingtimeandacceleratingdiscovery.
AlargeselectionofexpressionvectorscompatiblewiththeGatewayTechnologyisavailable,sonomatterwhatyourdownstreamanalysisneedsare,youcangettheremoreefficiently.
Gcontinuedonpage15table1-awidevarietyofGatewaydestinationvectorsareavailabletomeetyourdownstreamanalysisneedsunlimitedexpressionoptionsSuccessfulexpressionofagivengenefromaparticularpromoter,host,orsystemisnotguaranteed.
Togettheresultsyouneed,youmayhavetouseavarietyofvectorsandsystems.
WiththeGatewayTechnology,youcanaccessawidevarietyofexpressionandfunctionalanalysissystemswithoutadditionaltime-consumingsubcloningandsequencingsteps.
fastentryintoGatewayTechnologyAccesstounlimiteddownstreamcapabilitiesisrapidandefficientfollowinggenerationofaGatewayentryclone.
SimplycloneyourgeneofinterestintoaGatewayentryvector.
WhetheryoupreferTOPOCloning,PCRCloning,orrestrictiondigestionandligation,anoptionisavailableforyou.
Orsimplypurchaseapre-made,sequence-validatedopenreadingframe(ORF)thatisalreadyclonedintoanentryvectorbyorderingfromtheUltimateORFCloneCollection*.
easytouseOnceyouhaveyourgeneofinterestorORFinaGatewayentryvector,selectaGatewaydestinationvectorfromthewideselectionofavailable(table1).
Youcanquicklyandeasilyshuttleyourgeneofinterestfromtheentrycloneintooneormoredestinationvectors.
SimplyincubatetheentrycloneanddestinationvectorinaGatewayrecombinationreactionforonehouratroomtemperature.
ThentransformintoE.
coliandplate.
It'sthateasy.
tryGatewaytodayNomatterwhichsystemyouchoose,youwillgetresultsfasterwiththeflexibilityofGatewayTechnology.
FindoutmoreaboutGatewayatwww.
invitrogen.
com/gateway.
*ForalistofclonesavailableintheUltimateORFCloneCollection,visitwww.
invitrogen.
com/clones.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
VectorAdvantagesCat.
no.
High-level,cell-freeexpressionofactiveproteinpEXP1-DESTT7promoter,ribosome-bindingsite,andT7terminatorforefficientproteinsynthesisV960-01N-terminalXpresstagforconvenientdetectionoffusionproteins;tagcanberemovedfornativeexpressionN-terminal6xHissequenceforrapidpurificationpEXP2-DESTT7promoterandT7terminatorforefficientexpressionV960-02C-terminalV5-6xHistagforeasydetectionandrapidpurificationExpressioninE.
coliChampionpET-DEST42T7promoterforhighyieldsofrecombinantprotein12276-010LacOoperatorsequencesforlacrepressorbindingfortighterregulationLacrepressorfortightregulationoftranscriptioninE.
coliC-terminalV5-6xHistagforeasydetectionandrapidpurificationChampionpET104-DESTT7promoterforhighyieldsofrecombinantproteinK104-01N-terminalBioEasetagforinvivobiotinylationoffusionproteinsT7promoterundercontroloftheT7RNApolymeraseforhigh-levelexpressionpDEST14Notagfornativeproteinexpression(pDEST14)11801-016pDEST15,pDEST24GSTtagforeasycolumnpurification,pDEST15(N-terminal)andpDEST24(C-terminal)11802-014,12216-016pDEST17N-terminal6xHistagforrapidpurification(pDEST17)11803-012Expressions10.
4www.
invitrogen.
com15GatewayTechnologycontinuedfrompage14VectorAdvantagesCat.
no.
pBAD-DEST49araBADpromoterfortightlyregulatedexpressionoftoxicproteins12283-016N-terminalthioredoxinfusionpartnerforefficientproteintranslationandimprovedsolubilityC-terminalV5-6xHistagforeasydetectionandrapidpurificationOptimized,inducibleexpressioninS.
cerevisiaepYES-DEST52GAL1promoterforhigh-level,inducibleexpressioninS.
cerevisiae12286-019C-terminalV5-6xHistagforeasydetectionandrapidpurificationExpressionininsectcellsBaculoDirectDNABaculoDirectLinearDNAforeasiestmethodofbaculovirusproduction,idealforHTP12562-013Thymidinekinase(TK)genefornegativeselectionofnon-recombinantbaculovirusLacZgenetodetermineviralstockpurityC-terminalV5-6xHistagforeasydetectionandrapidpurificationpDEST8Bac-to-Bacvectorsforrapidbaculovirusproductionforsmallscaleexpression11804-010pDEST10Notagfornativeexpression(pDEST8)11806-015pDEST20N-terminal6xHistagforrapidpurification(pDEST10)11807-013N-terminalGSTtagforeasycolumnpurification(pDEST20)pMT-DEST48DESvector,aneasy-to-usesystemforconstitutiveorinducibleexpression12282-018Metallothioneinpromoterforhigh-level,metal-inducibleexpressioninDrosophilaS2cellsC-terminalV5-6xHistagforeasydetectionandrapidpurificationpMT/BioEase-DESTMetallothioneinpromoterforhigh-level,metal-inducibleexpressioninDrosophilaS2cellsV4140-20N-terminalBioEasetagforinvivobiotinylationoffusionproteinsExpressioninmammaliancellsCMVpromoterforhigh-levelexpressionmammaliancellsandGeneticinmarkerforrobustselection(exceptpcDNA6.
2/V5-DEST)pcDNA-DEST40C-terminalV5-6xHistagforeasydetectionandrapidpurification(pcDNA-DEST40)12274-015pcDNA-DEST47,pcDNA-DEST53GFPtagforrapidfluorescentdetectionofrecombinantprotein(pcDNA-DEST47(C-terminal)andpcDNA-DEST53(N-terminal))12281-010,12288-015pcDNA3.
2/V5-DESTC-terminalV5tagforeasydetection(pcDNA3.
2/V5-DESTandpcDNA6.
2/V5-DEST)12489-019pcDNA6.
2/V5-DESTBlasticidinresistancegeneforefficientstableselection(pcDNA6.
2/V5-DEST)12489-017pcDNA3.
1/nV5-DESTN-terminalV5tagforeasydetection(pcDNA3.
1/nV5-DEST)12290-010pDEST26N-terminal6xHistagforrapidpurification(pDEST26)11809-019pDEST27N-terminalGSTtagforeasycolumnpurification(pDEST27)11812-013CMV/TetO2promoterfortetracycline-regulatedexpressionandhighestlevelsofinducedexpressionpT-REx-DEST30Notagfornativeproteinexpression(pT-REx-DEST30)12301-016pT-REx-DEST31N-terminal6xHistagforrapidpurification(pT-REx-DEST31)12302-014EF-1αpromoter(forhigh-levelconstitutiveexpressionfromanon-viralpromoter)pEF-DEST51C-terminalV5-6xHistagforeasydetectionandrapidpurification12285-011pEF-FRT/V5-DESTFlprecombinasetarget(FRT)siteforefficientintegrationintoFlp-InCellLinesV6020-20HygromycinresistancegeneforconvenientselectionofintegrantsC-terminalV5tagforeasydetectionViraldeliverysystemspLenti6/V5-DESTCMVpromoterforhigh-levelexpressioninavarietyofmammaliancellsV496-10pLenti4/V5-DESTVSV-GenvelopeproteinfortransductioninabroadhostrangeV498-10C-terminalV5tagforeasydetectionChoiceofeitherBlasticidin(pLenti6/V5-DEST)orZeocin(pLenti4/V5-DEST)resistancegenesforefficientselectionandoptiontogeneratedoublestablecelllinespLenti6/UbC/V5-DESTHumanubiquitinpromoter(UbC)forinvivostudiesinanimalmodelsorforphysiologicalV499-10expressionlevelsinmammaliancellsC-terminalV5tagforeasydetectionpAd/CMV/V5-DESTCMVpromoterforhigh-levelexpressioninavarietyofmammaliancells(pAD/CMV/V5-DEST)orV493-20pAd/PL-DESTPromoterlessvectorforflexibilitytouseyourownpromoter(pAd/PL-DEST)V494-20E1andE3genesdeletedforproductionofreplication-incompetentvirusC-terminalV5tagforeasydetectiontable1-awidevarietyofGatewaydestinationvectorsareavailabletomeetyourdownstreamanalysisneeds,continued18009556288Expressions10.
4questionsanswerts16MagicMarkXPWesternProteinStandardHowcanWesternBreezeImmunodetectionKitsspeedupmyproteindetectionWesternBreezeistheonlycomplete,pre-optimizedwesternblottingsystemdesignedtoobtainsensitiveresultsinminimaltime.
Unlikeotherblottingkits,eachWesternBreezeKitincludesallthereagentsyouneedforblottinganddetectionwithyourownprimaryantibody.
Allkitreagentshavebeenoptimizedatapredeterminedconcentrationandtestedtoworktogethertoensuregreatresults.
There'snoneedforyoutospendtimeandeffortgathering,preparing,andtestingyourownsolutions.
WithWesternBreeze,alloftheoptimizationworkhasbeendoneforyou,savingyouhoursoftimeandeffort.
HowmuchtimecanIsavewithWesternBreezeThefastandsimpleWesternBreezeproto-colallowsyoutogetgreatwesternblotresultsinlessthanthreehours.
Thetimerequiredforallmajorsteps–includingblock-ing,primaryantibodyandsecondaryanti-bodyincubation–ismuchlessthanthatneededinconventionalprotocols(table1).
Inaddition,theWesternBreezeready-to-useandeasy-to-dilutereagentsminimizethetimespentpreparingsolutions.
Alltogether,you'llsaveupto17hourswithWesternBreezeKitsoverotherwesternblotdetectionmethods.
HowcanIvisualizethesignalsachievedwithWesternBreezeThealkalinephosphatase(AP)-basedWesternBreezeImmunodetectionKitsenableyoutoeasilyvisualizeyourproteinbandsusingeitherchemiluminescentlightemissionorcolorimetricstain.
TheWesternBreezeChemiluminescentKitsapplyaready-to-useCDP-StarsubstratethatiscatalyzedbyAPtoproducelightsig-nalsatthespecificproteinbands.
Thesesig-nalscanbecapturedbyX-rayfilm(figure1A),CCD-cameraimagesystems,andmostluminometers.
TheWesternBreezeChromogenicKitsuseaready-to-useBCIP/NBT*substratethatiscatalyzedbyAPtoformastablepurplecoloratthespecificbandsdirectlyonthemembrane(figure1B).
WithWesternBreeze,you'llbeabletoeasilyobtainwesternblotresultsbyfilm,imagesystem,orcolorimetricstain,whicheverissuitableforyourlaboratorysetting.
fastandeasyproteindetectionYoucansavetimeandeffortinbothopti-mizingsolutionsandobtainingfinalresultswiththeWesternBreezeImmunodetectionKits.
Sixkitsarecurrentlyavailablesoyoucanchoosetheonethatbestfitsyourneeds.
Toexperiencefastproteindetectionwithminimaleffort,callInvitrogenandplaceyourordertoday.
Ready-to-usewesternblottingkitsprovidefasterproteindetectionostcommerciallyavailablewesternblottingdetectionsystemsrequireyoutospendasubstantialamountoftimeoptimizingreagentsandprotocols.
Thepre-optimizedWesternBreezeImmunodetectionKitseliminatetediousoptimizationstepsandallowyoutoquicklyandeasilyobtainwesternblotresults.
You'llsavetimeandeffortoneverywesternblot.
MMajorStepsWesternBreezeProtocolConventionalProtocolPreparesolutions5minutes30minutesIncubatemembraneinblockingsolution30minutes1to15hoursIncubatemembraneinprimaryantibody1hour1to2hoursIncubatemembraneinsecondaryantibody30minutes1to2hoursTotaltimecomparisonoftheWesternBreezeandconventionalwesternblottingprotocolsfigure1-sensitivesignalsachievedwiththeWesternBreezeKitProductQuantityCat.
no.
PriceWesternBreezeChromogenicDetectionKitAnti-Mouse1kitWB7103$205Anti-Rabbit1kitWB7105$205Anti-Goat1kitWB7107$205WesternBreezeChemiluminescentDetectionKitAnti-Mouse1kitWB7104$255Anti-Rabbit1kitWB7106$255Anti-Goat1kitWB7108$255*BCIP/NBTisacompoundof5-bromo-4-chloro-3-indolyl-1-phosphate/nitrobluetetrazolium.
Eachkitsuppliessufficientreagentsfordetectionof20mini-blots.
OnenanogramofhumanIgGdevelopedwiththeWesternBreezeChemiluminescent(A)andChromogenic(B)Kits.
ABExpressions10.
4www.
invitrogen.
comproductreview17PCRSuperMixesconveniencePCRisanextremelyusefultoolthatisoftenmoretimeconsumingthanmanyuserswouldlike.
InvitrogenPCRSuperMixesaretheidealanswertothisdilemma.
Thesepre-mixedsolutionssaveyouvaluabletimebycombin-ingallofthePCRcomponents—enzyme,hot-startantibodies,dNTPs,magnesium,andbuffers—inonevial.
StrictqualitycontrolguidelinesensureproductconsistencytoimproveyourPCRresults.
WithPCRSuperMixes,you'llgettheconvenienceandreliablePCRperformanceyouneed.
high-qualityreagentsPCRSuperMixandPlatinumPCRSuperMixaredesignedforreliabilityandhighquality.
Toensurethatyoucontinuetoachievereli-ableresults,thequalitycontrolproceduresfortheseproductsarenowevenmorerigor-ous.
FunctionaltestingisdoneonmultiplevialsineverylotofeachSuperMixmanufac-tured.
Bandquality,consistency,andintensi-tyarevisualizedonanagarosegelfromthePCRdoneintriplicate.
TestsarealsodonetoconfirmtheabsenceofcontaminantssuchasDNase,RNase,andexonuclease.
Asaresultofthisextensivequalitycontroltest-ing,youwillgetreliablePCRperformance.
consistencyandpeaceofmindPCRSuperMixandPlatinumPCRSuperMixconsistentlyamplifytargetsupto5kb,withverylittlevariabilitybetweenlots(figure1).
Youcanhavecompleteconfidencethatthesetwohigh-performanceSuperMixesproduceyoureliableresultsthefirsttime,everytime.
selectiontomeetyourPCRneedsPCRSuperMixesareavailableinmultipleformatsforuseinallofyourPCRapplica-tions.
PCRSuperMixisforroutinePCR,offeringthecostsavingsofrecombinantTaqDNAPolymerasewiththeconvenienceofaSuperMix.
PlatinumPCRSuperMixincludesPlatinumhot-startantibodiesforimprovedPCRspecificityandyieldaswellasthecon-venienceofroomtemperaturereactionset-up.
AdditionalPCRSuperMixesareavailabletomeetallofyourPCRneeds.
TheseincludePlatinumPCRSuperMix96—allthebenefitsoftheoriginalPlatinumPCRSuperMixina96-wellformat.
CallorvisittheInvitrogenwebsiteforinformationonadditionalPCRSuperMixes.
ordertodayForreliable,convenientPCRperformance,choosePCRSuperMixandPlatinumPCRSuperMix.
Orderonetoday.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
ImprovedPCRSuperMixesofferqualityandconvenienceinoneousavevaluabletimewhenyouusePCRSuperMixesbecauseallofthereagentsyouneedforPCRarepre-mixed.
Nowyouwillalsohaveincreasedpeaceofmindbecausetheimprovedqualitycontrolproceduresguaranteehigherconsistency.
YProductQuantityCat.
no.
PCRSuperMix100reactions10572-0145000reactions10572-063PlatinumPCRSuperMix100reactions11306-0165000reactions11306-081PlatinumPCRSuperMix96skirted5x96-wellplates11306-065nonskirted5x96-wellplates11306-073figure1-SuperMixconsistencyfromlot-to-lotPCRreactionswererunwithfourdifferentgenomictemplatesranginginsizefrom670bpto4100bp.
ThreedifferentlotsofPCRSuperMixandPlatinumPCRSuperMixwereusedforeachofthegenomictargets.
Theselotswereruninduplicate.
Thegelwasloadedusing10%ofthe50-lPCRreaction.
Genomictemplates:Rhod670bpRhod1242bpRhod2579bpRhod4100bpLot11581752kb1kbLot1159371Lot1163000PlatinumPCRSuperMixPCRSuperMix2kb1kbLot1121640Lot1136038Lot115934818009556288Expressions10.
4productupdate18thecell-freeadvantageInvitro,cell-freeexpressionsystemstakelesstimeandeliminateseveralofthetime-consumingstepsrequiredbycell-basedexpressionsystemssuchastransformation,transfection,andmaintenanceofcellcul-tures.
Althoughtherearesignificantadvan-tagestousingacell-freeexpressionsystem,thesemethodsoftenproduceextremelylowamountsofprotein.
WiththeExpresswayPlusSystem,youcanscaleyourexpressiontosuityourdownstreamneeds.
Thereac-tionisscalablefrommicrogramstomil-ligramsofproteindependinguponthereac-tionsize.
Whicheveryouchoose,you'llhavetheproteinyieldyouneedinasimpletwo-hourreaction.
activeproteinmadeeasyTheExpresswayPlusSystemisasimple-to-use,cell-freeexpressionsystem.
Simplycombinethereactionbuffer,methionine,andT7RNApolymerasewiththehighlyactiveE.
coliextract.
ThenaddyourDNAtemplatecontainingaT7promoter,ribo-somebindingsite(RBS),andyourgeneofinterest.
Thereaction-drivingE.
coliextractquicklytranscribesandtranslatesactivepro-teinfromtheDNAtemplate(figure1).
TheExpresswayPlusreactionrequiresonlytwohourstocomplete,savingyouhoursoftimeovertraditionalexpressionmethods.
scalablereactionTodemonstratethescalabilityoftheExpresswayPlusSystem,reactionvolumesrangingfrom50lto10mlwerecompared.
Followingatwo-hourincubation,proteinyieldsfromeachreactionwerealsodeter-mined(figure2).
Increasingthereactionvolumeresultedinasubsequentincreaseinproteinyield.
UsingtheExpresswayPlusSystem,youcanproducemilligramsofpro-teinsandgettoyourdownstreamexperi-mentsfaster.
moreprotein,fastandeasySpeedupyourproteinproductionandgetyourresultsfasterwiththeExpresswayPlusExpressionSystem.
Callandordertoday.
*EachkitincludesIVPSPlusE.
coliExtract,2.
5XIVPSPlusE.
coliReactionBuffer,RNaseA,T7EnzymeMix,Methionine,reactiontubes,controlplasmids,andtheappropriateexpres-sionvectorwhenindicated.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
Makeactiveproteininascalable,two-hourcell-freereactionExpresswayPlusExpressionSystemheExpresswayPlusExpressionSystemutilizesanefficientcoupledtranscriptionandtranslationreactiontoproduceactiverecombinantprotein.
Injusttwohours,youcanproduceuptomilligramsofproteinsuitableforanarrayofdownstreamapplications.
TProductQuantityCat.
no.
ExpresswayPlusExpressionSystem*withoutvector20reactionsK9900-01withpEXP1-DEST20reactionsK9900-02withpEXP2-DEST20reactionsK9900-03figure1-howtheExpresswayPlusSystemworks5T7RNAPolymerasePlasmidorLinearDNAProteinRibosomes3EnergyRenewalSystemAsaDNAtemplatedrivenbyaT7promoteristranscribed,the5endofthemRNAisboundbyribosomesandundergoestransla-tion.
AspeciallyengineeredATPenergyrenewalsystemcoupledwithearlyribosomebindingfortranscriptstabilityresultsinhighproteinyields.
figure2-scale-upofproteinexpressionusingtheExpresswayPlusSystemTheincreaseinExpresswayPlusreactionvolumeresultsinincreasedβ-galactosidaseproteinyields.
yield(g)ReactionVolume010001500200025003000350050050l250l500l1ml5ml10mlExpressions10.
4www.
invitrogen.
comnewproduct19I-SAGELongKitimprovedexpressionprofilingThoughpowerfulintheirabilitytosimulta-neouslymonitorthousandsofdifferentiallyexpressedgenes,microarrayslimitexpressionprofilingtothenumberofprobesattachedtotheirsurfaceandrequiregenesequenceknowledge.
Moreover,variationsinsamplepreparation,slidepreparation,hybridization,andnormalizationtechniquesmakedatacomparisonsbetweenmicroarrayexperi-mentsdifficult.
SAGEcircumventstheselimitations,allowingyoutomonitorgeneexpressionofeverytranscriptinacell,gener-atequantitativedata,andeffectivelydetectlowabundancetranscripts.
ThenewI-SAGELongKitoffersallthebenefitsofSAGEandmore.
Usinganimprovedprotocol,theI-SAGELongKitgeneratesalongertagthanispossiblewiththeoriginalSAGEmethods,allowingclearerdiscriminationbetweengenesandimprovingnovelgeneisolation.
increasedaccuracyTheoriginalSAGEmethodisolatesa14-bptagfromeachmRNAtranscriptexpressedinasample.
Tagsareligated,cloned,andsequencedinhighthroughput.
RawsequencedataisanalyzedusingSAGEdataanalysissoftware,producingdigitalresultsdetailingthepresenceandcorrespondingfrequencyofmRNAtranscripts(1).
TheI-SAGELongKitusesasimilarprotocol,butwithanewrestrictionendonuclease,MMEI,producinga21-bptag.
Theaddition-altaglengthenhancestheidentificationofmRNAtranscripts,enablingmoreaccuratecomparisonstoreferenceESTdatabases(2).
enhancedownstreamstudiesTheadditionallengthofLongSAGEtagsprovidessufficientcomplexitytoaccuratelymaptagsdirectlytotheirgenomiclocation,enablingyoutomatchdifferentiallyexpressedtranscriptstotheirchromosomallocation(table1).
You'llbeablecorrelateincreasesordecreasesingeneexpressionwithbiologicalfunctionandidentifydiseaserelatedgenes.
AftercomparingyourI-SAGELonglibrariestoreferenceESTdatabasesinSAGEmaporSAGEGenie*,youcanuseuniqueLongSAGEtagstodesignPCRprimersforuseinRapidAmplificationofcDNAends(RACE)applicationsorasprobestoscreencDNAlibraries,facilitatingnovelgeneisolation(2).
completekitsavestimeSourcingandoptimizingthenearly50reagentsnecessarytocreateLongSAGElibrariesisnotnecessary.
TheI-SAGELongKitcontainsalltherequiredreagentstosuc-cessfullygenerateLongSAGElibrariesinonefunctionallytestedkit.
improveyourSAGEstudiestodayTheI-SAGELongKitenablesyoutoenjoythebenefitsofLongSAGEandimproveyourexpressionprofiling.
Callandordertoday.
*DatabaseURLs:SAGEGenie:http://cgap.
nci.
nih.
gov/SAGE.
SAGEmap:http://www.
ncbi.
nlm.
nih.
gov/SAGE.
References:1.
Velculescu,V.
etal.
(1995)Science270:484-487.
2.
Saha,S.
etal.
(2002)NatureBiotechnology19:508-512.
TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(SeetheInvitrogencatalogorwebsite,www.
invitrogen.
com).
BytheuseoftheseproductsyouacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.
ExpandyourexpressionprofilingstudiesheI-SAGELongKitimprovesthewidelyreferencedserialanalysisofgeneexpression(SAGE)method,enablingmoreaccurateESTdatabasecomparisonsanddirectmappingoftagstogenomiclocation.
TheI-SAGELongKitisanidealcomplementtomicroarrayanaly-sisoranexcellentalternativeforresearchersseekinganeffectivemethodofexpressionprofil-ingwithoutthelimitationsofmicroarraytechnology.
TProductQuantityCat.
no.
I-SAGELongKit5LibrariesT5000-03I-SAGELongDitagPCRModule1,000PCRrxnsT5000-04InvitrogenMagneticStand1R670-01table1-theoreticalmatchingoftagstogenomeTaglengthTaguniqueness(nbasepairs)Complexity*probability141,048,5760.
00%154,194,3040.
08%1616,777,21616.
73%1867,108,86463.
95%191,073,741,82497.
24%204,294,967,29699.
30%2117,179,869,18499.
83%*Complexityoftags(C=4(n-4))isdeterminedusingataglengthcompris-ingaconstant4bprepresentingtherestrictionsiteatwhichthetran-scriptwascleaved,followedbynbpderivedfromtheadjacentsequenceineachtranscript.
Theprobabilitythatatagisuniqueinthegenome(Pu=[C-1/C]30,000,000)isdeterminedundertheassumptionthatthegenomecontains~30x106NlaIII-derivedtagsandiscomprisedofrandomsequence.
PRSRTSTDU.
S.
PostagePAIDPermitNo.
825SanDiego,CACorporateheadquarters:InvitrogenCorporation1600FaradayAvenueCarlsbad,CA92008USATel:7606037200Fax:7606026500TollFreeTel:8009556288TechnicalService:8009556288ext.
2E-mail:tech_service@invitrogen.
comwww.
invitrogen.
comEuropeanheadquarters:InvitrogenLtdInchinnanBusinessPark3FountainDrivePaisleyPA49RF,UKTel:+44(0)1418146100Fax:+44(0)1418146260E-mail:eurotech@invitrogen.
comBaculoDirect,BL21Star,BioEase,Cellfectin,Champion,Clonase,DES,DEST,DH5α,EvoQuest,Expressway,Flp-In,Gateway,Geneticin,I-SAGE,Lipofectamine,LUX,Mach1,Mark12,MAXEfficiency,MultiShot,NuPAGE,OmniMAX,OneShot,pcDNA,pENTR,Platinum,Ready-Load,SimplyBlue,SuperScript,SureLock,Tag-On-Demand,TOPO,Ultimate,Verve,ViraPower,Vivid,WesternBreeze,Xpress,Z-LYTE.
editorRobynLeungauthorsLouisGerrueRobynLeungScottMogullJohnHudsonKerryLowrieMarisaPearcePeterJozsiTinaMaiJillWinerCarolBethLandSandeepMehtaproductiongraphicmanagerGerriHallingdesignerVinhNguyenPrintedonRecycledPaperPrintedintheU.
S.
A.
2003InvitrogenCorporation.
Allrightsreserved.
Reproductionforbiddenwithoutpermission.
PricesareinU.
S.
dollarsandsubjecttochange.
contentsvolume10issue4newproductsBaculoDirectBaculovirusExpressionSystemFreeyourhandswithsimplifiedbaculovirusexpression2SuperScriptIndirectcDNALabelingSystemIncreasemicroarraysensitivity3Mach1T1RandOmniMAXT1RChemicallyCompetentE.
coliClonegenesfasterandmoreefficiently.
.
.
.
8SuperScriptIIIOne-StepRT-PCRSystemsforEndPointandReal-timeAnalysisHigh-performanceone-stepRT-PCRforendpointandreal-timeapplications.
.
.
9Tag-On-DemandTechnologyFlexibleproteinexpression—getnativeortaggedproteinondemand10UltimateMouseORFClonesSpeedupyourresearchwithready-to-goclones12ExpresswayPlusExpressionSystemMakeactiveproteininascalable,two-hourcell-freereaction18I-SAGELongKitExpandyourexpressionprofilingstudies.
.
19applicationnoteSimplyBlueSafeStainSimplyBlueSafeStainoutperformsthecompetitioninproteinstaining6productreviewsVerveMammalianTwo-HybridKitwithTOPOToolsTechnologyRapidandaccuratemammaliantwo-hybridanalysis4ChampionpETExpressionSystemThefastestwaytogeneratehigh-levelpETexpressionconstructs5EvoQuestCustomPeptidesandAntibodiesEasyon-lineordering13GatewayTechnologyComprehensiveselectionofGatewayexpressionvectors14PCRSuperMixImprovedPCRSuperMixesofferqualityandconvenienceinone17announcementInvitrogenacquiresPanVera13Q&AWesternBreezeImmunodetectionKitsReady-to-usewesternblottingkitsprovidefasterproteindetection16ExpressionsispublishedbyInvitrogenCorporation.
ThefollowingaretrademarksofInvitrogenCorporation:CDP-StarisaregisteredtrademarkofTropix,Inc.
CoomassieisaregisteredtrademarkofImperialChemicalIndustries,PLC.
GelCodeisaregisteredtrademarkofPierceChemicalCompany.
PrismisaregisteredtrademarkofAppliedBiosystems.
SAGEisatrademarkofGenzymeCorporation.
XL10-GoldisaregisteredtrademarkofStratagene.
ZeocinisatrademarkofCAYLA.
320-022122051203ACD