Description
Basedonourproprietarypolymersynthesistechnology,PolyJetDNAInVitroTransfectionReagentisformulatedasabiodegradablepolymerbasedDNAtransfectionreagentthatensureseffectiveandreproducIBLetransfectiononHEK293,COS-7,NIH-3T3,HeLa,CHOandabroadrangesofhard-to-transfectmammaliancells.PolyJetreagentisabletoimmobilizeDNAmigrationduringelectrophoresisatverylowconcentrationandformtransfectioncomplexwithin5minutesatRT.Aremarkablefeatureofthereagentistherapidandcompletedegradationofpolymeraftertransfectioncomplexendocytosis(Figure1),leADIngtomuchlesscytotoxicity.PolyJetreagent,1.0ml,issufficientfor~667transfectionsin24wellplatesor~333transfectionsin6wellplates,providingaveryaffordablealternativetotheleadingproductsfortransfectingavarietyofcommonlyusedandhard-to-transfectmammaliancells.
Figure1.ACartoonShowingBiodegradationofPolyJetDNATransfectionReagentAfterEndocytosisofTransfectionComplex
Features
-Bio-degradableafterendocytosis
-Exceptionalhightitersofvirusproduction
-EquallygoodforverylongDNAs(>89kb)
-EquallygoodforbothsingleDNAtransfectionandmultiDNAco-transfection
-Highlevelsofrecombinantproteinproduction
-Simple&robusttransfectionprocedure
-Veryaffordable
StorageCondition
Storeat4°C.Ifstoredproperly,theproductisstablefor12monthsorlonger.
BroadTransfectionSpectrumforMammalianCellTypes
CellLines | Efficiency(%GFP) | CellLines | Efficiency(%GFP) |
| McArdle7777 Hep3D SHEP 3T3-442A COS-7 CV-1 D407 DHDPro.b 3LL B16-F10 BAEC BHK-21 CaSki CaCo2 CHO HCS-2/8 HEK-293 HeLa HLMEC H-MVEC Huh-7D12 ATT20 SK-N-SH C2C12 HepG2 | 65-70% | hESCs | 70% |
ExamplesShowingTransfectionEfficiencyofPolyJetDNAInVitroTransfectionReagentonCommonlyUsedCells
TransfectionefficiencycomparisonofPolyJetvs.lipofectaminePlusonChineseHamsterOvary(CHO)cells.HAtaggedbeta-tubulinCDNAwasdeliveredintoCHOcellswithPolyJet(leftpanel)andlipofectaminePlus(rightpanel)respectively.FITCconjugatedantibodyagainstHAtagwasutilizedtopickupHA-beta-tubulin(Green)whileaDM1aantibodywasusedtodetectendogenousalpha-tubulinfollowedbyprobingwithrhodamineconjugatedsecondaryantibody(Red).TheabovepicturewasprovidedbyDr.ShangYinofUniversityofTexasatHoustonMedicalSchoolascourtesy
AcomparisonshowingtransfectionefficiencyofPolyJetreagentvs.aleadingproduct,Lipofectamine2000onHEK293FTcells.HEK-293FTcellsweretransfectedwithGFPvector(pEGFP-N3)byPolyJet(leftpanel)andLipofectamine2000(rightpanel)respectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope24hoursposttransfection
AcomparisonshowingtransfectionefficiencyofPolyJetreagentvs.aleadingproduct,Lipofectamine2000onHepG2cells.HepG2cellsweretransfectedwithGFPvector(pEGFP-N3)byPolyJet(leftpanel)andLipofectamine2000(rightpanel)respectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope24hoursposttransfection
TransfectionefficiencycomparisonofPolyJetvs.FugeneHDonMDCKcells.AplainGFPDNAwastransducedintoMDCKcellswithPolyJet(leftpanel)andFugeneHD(rightpanel)reagentsrespectivelypermanufacturers"protocols.GFPandDAPIstainingwerevisualizedunderfluorescencemicroscopy48hoursposttansfection.TheabovecomparisondataandpictureswerecompletedandprovidedbyDr.GeZhouofNYUMedicalCenterascourtesy
AcomparisonshowingtransfectionefficiencyofPolyJetreagentvs.aleadingproduct,Lipofectamine2000onMDCKcells.MDCKcellsarenotoriouslyhardtotransfect.Withproprietary"ShavedCellTransfection"protocol,PolyJet(leftpanel)givesupto70%GFPpositivecellsvs.Lipofectamine2000(rightpanel)around5%efficiency.MDCKcellsweretransfectedwithGFPvector(pEGFP-N3)byPolyJet(leftpanel)andLipofectamine2000(rightpanel)respectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope36hoursposttransfection
AcomparisonshowingtransfectionefficiencyofPolyJetreagentvs.aleadingproduct,FugeneHDonLNCapcells.LNCapcellsweretransfectedwithGFPvector(pEGFP-N3)byPolyJet(leftpanel)andFugeneHD(rightpanel)respectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope24hoursposttransfection
AimageshowingexceptionaltransfectionefficiencyofPolyJetreagentonHumanembryonicstemcells (hESCs). ThehESCsgrowninE8mediumonGeltrexvs(leftpanel,DICimaging)wastransfectedwithpEF1α-GFP.TheGFPexpression(rightpanel)wasvisualizedbyNikonEclipseFluorescencemicroscope24hoursposttransfection. TheabovepictureswereprovidedbyDr.MarinaPryzhkovaofJohnsHopkinsUniversityascourtesy
Neuro2AcellstransfectedwithpEGFP-C1plasmidusingPolyJetInVitroDNATransfectionReagent.TheNeuro2AcellswerevisualizedbyNikonEclipseFluorescencemicroscopewithDICphaseimaging(left)andFITCimaging(right)24hourspost-transfection
ComparisonofcytotoxicityofPolyJetDNAInVitroTransfectionReagentwithL2Konprimarymurineskinfibroblast.Theprimarymurinefibroblastwasincubatedwiththeindicatedtransfectionreagents/pEGFP-C1(DNA)complexesabovefor4hoursinserum-freeDMEMHighGlucosemediumfollowedbyreplacementofcompleteserum-containingmedium.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscopewithDICphaseimaging24hoursposttransfection
DataSheetandProtocols
-GeneralProtocolforTransfectingMammalianCells
-AShortProtocolforTransfectingMammalianCells
-AdvancedProtocolforTransfectingHard-to-TransfectMammalianCells
-AProtocolforTransfectingSUSPension293andCHOCells
-ProtocolforLentivirusProduction
-ProtocolforrAAVProduction
- TechnicalNote&TransfectionTips
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Testimonials:
Sorryforsooobigdelay,butnowIamtotallyinlovewithPolyJet.IhaveusedPolyJetforawhileonmouseESCswithrelativelygoodefficiency50~70%.
--------MarinaPryzhkova,Ph.D.,JHU
PolyJetTransfectionReagentworkedequallyaswellaslipofectamine2000,withlittleevidenceofcelldeathon293,PC-3and22RV1cells.Iwilldefiantlyconsiderswitchingover.
-----TiffanyWallace,Ph.D.,NCI/NIH
Ionlydidsidebysidewiththetestingsample(PolyJet)andLipo2000withGFPtransfectiononCOS-7cells.Theresultwasverygood.PolyJetwasevenbetterthanL2K.
------FengQiao,Ph.D.,NEI/NIH
ItestedthesampleofPolyJetonmyNIH-3T3mousefibroblaststhisweekend.TheresultsweremuchbetterthanLipofecatmineLTX.I"mattachingapowerpointslidewithmyresults(Ididnotquantifythe%transfectionefficiency,butthepicturesgetthepointacross).Ifoundthattheprotocolfordifficult-to-transfectcelllinesworkedbetterthanthestandardprotocol.
-----StephanieMurphy,Ph.D.,DartmouthCollege
Ihadchancetotryyourproductfinally.Itwasgreatsuccess.IusedHeLacellsandgot10%transfectionefficiency(<0.1%forLipofectamine).Thankyou!IwaswonderingifIalsotryGenJetPlusDNAInVitroTransfectionReagent?Accordingtoyourwebsite,thereagentworksbetterthanregularPolyJet.
-------YumiUetake,Ph.D.,UMASS
ItestedPolyJetanditlooksgreatonMDCK.Weplacedorder.Thankyou!
-------GeZhou,Ph.D.,NYU
Wearehappytoprovidefeedback.PolyJetworkedverywellforusinHepG2cells,wegotapproximately80%efficiencywithpMAXGFPplasmid,byfollowingtheconditionsinyoursuggestedprotocol.WeranacomparisonwithLipofectamine,whichonlyshowedapproximately20-30%transfectionefficiency.Weareplanningexperimentsandwillbeorderingmoresoon.
-------EmilyMcallister,PBRC
ebiomall.com
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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。
