Description:
Basedonourinnovativeandproprietarylipid-conjugationtechnology,LipoJet™TransfectionKit,formulatedfromnovelfluorinatedcationiclipids,exhibitssignificantdifferencefromotherlipidstransfectionreagentsinthemarket.LipoJet™TransfectionKitisthemostpowerfulyetverygentlegenedeliverytoolforavarietyofapplicationsincludingplasmidDNAand/or siRNAformostofmammaliancelltypes. ComparedwithleADIngproductsinthemarket, LipoJet™ismore cost-effectiveandalways provideshighertransfectionefficiencywithlesscytotoxicity.
Kitcontent:
-LipoJet™Reagent,1.0mlsufficientfor1000DNAtransfectionsand1000siRNAtransfections (24-wellplate).
-LipoJet™TransfectionBuffer(5x),8.0mLtomake40mLofworkingsolution(1x).
Features:
-Versatile:DNAtransfection,siRNAtransfectionandDNA/siRNAco-transfection.
-Powerful: Outstandingefficiencyonadherentcells.
-Extremelylowtoxicity: ImproveCellviABIlityaftertransfection
-Economical:Moretransfectionswithlessreagent
-Easytouse:OnetubereactionandcompatIBLewithserum/antibiotics.
-Suitableforhigh-throughputapplication
Storage:
Storeat4°CforLipoJetReagentandRTforLipoJetTransfectionBuffer(5x). Ifstoredproperly,theproductisstablefor12monthsorlonger.
BroadTransfectionSpectrumforMammalianCellTypes
CellLine | DNATransfection | siRNATransfection |
293,293T | 85% | - |
ExamplesShowingExcellentDNAandsiRNATransfectionEfficiencyofLipoJet™InVitroTransfectionKit
AefficiencycomparisonofLipoJet™reagentvs.brandnameproductstotransfectHela,Cos-7,NIH-3T3,CHOandHEK293(leftpanel)andCos-7(rightpanel). pEGFP-N3CDNA(0.5µg/wellof24-wellplate)wastransfectedintodifferentmammaliancellsperthestandardtransfectionprotocolsinpresenceofserum(10%FBS)andantibioticsasrecommendedbymanufacturers. ThetransfectionefficiencywereanalyzedviaFACS48hoursposttransfection.
AtoxicitycomparisonofLipoJet™reagentvs.brandnameproductstotransfectHelacells. pEGFP-N3cDNA(1.0µg/wellof24-wellplate)wastransfectedtoHelacellsperthestandardtransfectionprotocolsinpresenceofserum(10%FBS)andantibioticsasrecommendedbymanufacturers. TheMTTassay(rightpanel)andphasecontrastimaging(leftpanel)wereusedtoanalyzethecellviability48hoursposttransfection.
AcomparisonofLipoJet™reagentvs.Lipofectamine2000(L2K)andPolyJet™transfectionreagentonHEK293Tcell.pEGFP-N3cDNA(0.125µg/well,0.25µg/welland0.5µg/perwellof24-wellplate)wastransfectedinto293Tcellsusingthestandardtransfectionprotocolsinpresenceofserum(10%FBS)withLipoJet™(upperpanelatLipoJet™/DNA(µl/µg)ratio=2),L2K(middlepanelatL2K/DNA(µl/µg)ratio=3)andPolyJet™(lowerpanelatatPolyJet™/DNA(µl/µg)ratio=3)respectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope48hoursposttransfection.
AcomparisonofLipoJet™reagentvs.Lipofectamine2000(L2K)andPolyJet™transfectionreagentonHelacell. pEGFP-N3cDNA(0.125µg/well,0.25µg/welland0.5µg/perwellof24-wellplate)wastransfectedintoHelacellsusingthestandardtransfectionprotocolsinpresenceofserum(10%FBS)withLipoJet™(upperpanelatLipoJet™/DNA(µl/µg)ratio=2),L2K(middlepanelatL2K/DNA(µl/µg)ratio=3)andPolyJet™(lowerpanelatatPolyJet™/DNA(µl/µg)ratio=3)respectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope48hoursposttransfection.
ExceptionalgenesilencingofLipoJet™reagentduringDNA/siRNAco-transfectiononmammaliancells.TransfectionofGFPcDNA(leftpanel,0.25µgperwellof24-wellplate)andco-transfectionofGFPcDNA(0.25µgperwellof24-wellplate)andGFPtargetedsiRNA(final10nM,rightpanel)withLipoJet™reagentleadstoexceptionalgenesilencingonHEK293(upperpanel),Hela(middlepanel)andSaoS-2(lowerpanel)cellsrespectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope48hoursposttransfection.
ExceptionalDNA/siRNAco-transfectionefficiencyonHUVEC.Co-transfectionofmCherrycDNA(0.10µgperwellof24-wellplate)andFITCconjugatedsiRNA(final30nMperwellof24-wellplate)toHUVECwithLipoJet™reagentgaveriseto60%mCherry+(phasecontrastoverlappedwithmCherryimaging,leftpanel)andnearly100%FITC-siRNA+(phasecontrastoverlappedwithFITCimaging,rightpanel)HUVEC24hoursaftertransfection. ThepicturesweregivenfromDr.PanKongofUSCascourtesy. 
ExcellentsilencingofendogenouslyexpressedKIF11(alsoknownasEG5)inHEK293(upperpanel)andHela(lowerpanel)cellswithLipoJet™reagentat10nMEG5siRNA.KIF11(alsoknownasEG5)encodesamotorproteinthatbelongstothekinesin-likeproteinfamilyinvolvedinchromosomepositioningandbipolarspindleformationduringcellmitosis.AreductioninKIF11levelscausesmitoticarrest.LipoJet™reagenteffectivelydeliversEG5siRNA(final10nM)toHEK293andHelacells,leadingtomorethan80%of"round-up"phenotypeofHEK293andHelacells24hposttransfectionovernegativecontrol(final10nMwithshamEG5siRNA).Thephenotypeof"rounded-up"HEK293andHelacellswerevisualized24hposttransfectionwithaNikonmicroscope.
AimageshowingexceptionaltransfectionefficiencyofLipoJetTransfectionKitonHumanembryonicstemcells (hESCs). ThehESCsgrowninE8mediumonGeltrexvs(leftpanel,DICimaging)wastransfectedwithpEF1α-GFP.TheGFPexpression(rightpanel)wasvisualizedbyNikonEclipseFluorescencemicroscope24hoursposttransfection.TheabovepictureswereprovidedbyDr.MarinaPryzhkovaofJohnsHopkinsUniversityascourtesy
DataSheet&Protocol:
-AProtocolforDNAandsiRNATransfection
-AShortProtocolforDNATransfection
-AShortProtocolforsiRNATransfection
-AProtocolforDNA/siRNACo-transfection
- TechnicalNote&TransfectionTips
Weareoffering"WirelessPowerPointPresenter"tocustomerswhopurchase5x1.0mLLipoJet™reagentbyDecember2014.Takeadvantageofitanddon"tletthischancego...
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FG-3:WirelessPowerPointPresenterwithCaseforpurchaseof5X1.0mlormore.Thispresenterisofferedforlimitedtimeonly! |
Torequestafreetrialsample,pleaseCreateAnAccountwithustoenteryourshippingaddressandemailusatorder@Signagen.com
Testimonials:
Sorryforsooobigdelay,butnowIamtotallyinlovewithbothPolyJetandLipoJet.RecentlywediscoveredthatLipoJethassupergoodtransfectionefficiencywithmESCsandhESCswithi muchlesstoxicity.LastweekItransfectedovernighthumanESClineH1usingLipoJetwithsameamazinglyhighefficiency-upto50~70%.DNAconstructincludesGFPunderEF1apromoter(CMVpromoterdoesnotworkinhESCs).ThisisthebesttransfectionefficiencyIeversawwithESCs.Lipofectamin3000didn"tgivemetheseresults(usedonmouseESCs).
--------MarinaPryzhkova,Ph.D.,JHU
Itriedtheplasmid/siRNAco-transfectionusingtheLipoJetsampleinEAhy926cellwhichisaHUVECcellline.Theresultsareprettygood,atleastforthetransfectionefficiency.Fortheplasmidtransfection,48hourslater,around60%cellaretransduced.ForsiRNA,theefficiencyisalmost100%.WeareorderingmoreLipoJetreagenttousefromnow.
-----Dr.PanKong,UniversityofSouthernCalifornia
Wegot>90%efficiencywithLipoJetvs.80%withX-tremeGENE9on293Tcell.Definitelywillordermore....
-----Dr.NuoYangfromRoswellParkCancerInstitute
HerearetheresultsfromourpreliminaryexperimentswithLipoJet.Weareverysatisfiedwithitsefficiency.Unfortunately,wewereoutofFugene6/HDandwereunabletotestthemsidebyside,butbasedonpreviousexperience,wedobelievethatyourproductworkedwithbetterefficiency.
------Dr.AlisonMcKelveyfromUniversityofPittsburgh
IamhappytosaythatIhadgreatsuccesswithyourtransfectionreagentsonOKF6/TERT2humankeratinocytes.IwillputtogetherthedataonceIhavecompletedmyanalysis.IreallyliketheLipoJet.MycellofinterestdidnotdoverywellintheGenMutealthoughmycontrolcellsdid.WewanttogoaheadwithorderingASAP.Ialsohaveacouponfor10%foraPOorder.
-----Dr.MichelleSimpson-AbelsonfromUPMC
WedidindeedtestthemsidebysidewithourhomemadeCaPhos.Theresultsaregreat, LipoJetandCalFectinbothdidextremelywell.LipoJetbeingthebetterofthetwo.Iwillsendyoutheresultswhen Igetthem, Iamcurrentlyoutofthelabbutwewillbeorderingmoreforcertain.Thankyouforsendingthosesamples.
------Dr.AhmedHassib,CornellUniversity
FortheLipoJet,IonlycompareditwithLipoLTXinHelacells.TheLipoJetgaveamuchhigherefficiency(~35%betterthanLipoLTX)24hoursaftertransfection.WewillordersomeLipoJet.
-----AbetatesterfromUSC
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其次要看下你选择单位的规模如何，做的比较好的，还是上海这边的，你可以看下基尔顿生物，原代细胞培养，动物造模，整体课题外包。
不建议你用MDCK细胞，非常难转染。
GFP发出绿色荧光的原理是Ca离子进入GFP的beta-barrel结构中引起的特定能级，因此只要这个结构仍然保持着，就可以发出荧光。
由于GFP的beta-barrel结构非常稳定，一些版本的GFP蛋白（如EGFP）甚至能抵抗94C的高温几分钟而不完全变性，因此想在溶液状态下去掉GFP的荧光是很难的，一般需要用光漂白法。
基于其非常稳定的结构，即便细胞被固定了，仍然会有一部分的GFP蛋白保持其构象而发出荧光。此时荧光可能较弱。在荧光显微镜下是有可能看得到的。
我刚开始做转染，悬浮细胞，分别做过表达和敲减，看了很多文献，大都没有提及转染后是用转染的这同一批细胞同时做pcr，wb，cck8，凋亡，细胞周期；还是说这次转染只做pcr或wb，再转染一次做cck8或细胞周期。剩下的功能试验均同前，转染一次做一次？我养的是悬浮细胞，转染后做cck8这些功能试验前需要离心换液吗？跪谢解答！
可以用移液器将细胞吸出来并高速离心，沉淀重悬于PBS中洗涤，接着就可以裂解提取蛋白了。可以用超声，酶解等等，裂解后离心收集上清。
其次要看下你选择单位的规模如何，上海这边的，你可以看下基尔顿生物，原代细胞培养，动物造模，整体课题外包。
实验室一直都是用日常型质粒抽提试剂盒，转染细胞没问题。
我觉得只要是注意以下2点就可以了：
1，注意大肠杆菌(Escherichia coli)本身的污染，收集菌体沉淀时防止菌液散落，经常用75%的乙醇擦拭手套。
2，最后洗脱时最好使用无内毒素的水，我们是用注射用水的。
无论是小提还是大提我们都是用的日常型的，并没有刻意用转染级的，因为转染量大，去内毒素的操作太麻烦，损失太大。
转染分2种，一种是瞬时转染，即转染后让细胞表达目的蛋白后即提取蛋白，提一次蛋白，转染一次，这种方式一般不传代；
另一种转染为稳定转染，转染后加入一定选择压力进行筛选，没有转染的细胞不能存活，只留下转染的细胞，这种情况下可以筛选单个转染细胞，构建稳定表达某一特定蛋白或基因的细胞系。
