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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DXY721认为：
悬浮细胞和贴壁细胞在转染过程中差别不大，主要差别在于转染后的筛选，当然如果你做的是瞬时转染就不存在筛选的问题了。
其实转染的过程很简单，问题是能不能转的进去的，转染率能有多少，转进去是否可以稳定表达目的蛋白等等。
我们也是用脂质体做悬浮细胞的转染，说明书上都有具体的操作过程，将脂质体和目的基因按比例混合，然后加到细胞悬液里就OK了，说的简单，实际上还是有一些细节要注意的，比如脂质体和目的基因混合的比例，转染的细胞数，细胞的代数，细胞的状态，有的还要求在转染的前一天传代一次，不过不要怕，这些在脂质体说明书上都有明确的说明，按照说明书做就可以了。
jinghuanlv认为：
悬浮细胞和贴壁细胞转染还是有很大不同的。
脂质体转染的原理基于电荷吸引原理，先形成脂质体-DNA复合物，散布在细胞周围，然后通过细胞的内吞作用，将目的基因导入细胞内，而脂质体复合物与贴壁细胞的接触机会比悬浮细胞高出很多倍，所以，脂质体转染时悬浮细胞的转染效率要明显低于贴壁细胞。
我们实验室转染悬浮细胞是用的电穿孔法，目前为止，悬浮细胞转染的最好方法还是电转，我们实验室用的电转仪是Bio-Rad的，使用条件是电压250V，电容975uF，效果不错，不妨一用。
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。
本人研究生
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。
各位版友求助，
我使用Hek293构建转染模型，瞬转5质粒，用lipo2000做转染体系。
转染48h，发现荧光较强的细胞都在爬片的边缘，比例十分少。是因为我添加试剂的手法不对吗。
同时也发现加入转染体系后细胞状态特别差。想问一下用lipo2000时可以用无双抗的10%FBSDMEM吗。我转染前6h现在用的是纯DMEM，不含FBS。
请各位大神帮忙
但是有在转染前，将细胞进行重新传代的情况，这样做的目的在于保持细胞的活性状态。
“转染前将细胞以1.5-4.5X104 cells/well 的量(具体接细胞数请参考表1）接种在孔板中，于37℃， 5%CO2 的条件下进行培养，18-24小时（ sf9细胞为3-4小时）后转染。”
