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Supplemental materials.doc
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SupplementalmaterialsMaterialsandMethodsConstructionofheNOSplasmidandadenovirusproductionThePMSCV-heNOSplasmidwasgenerouslypresentedbyProf.DelingKongfromNankaiUniversity.heNOSgenewasamplifiedfromthePMSCV-heNOSplasmidusingspecificprimerscontainingEcoRIandSalIsites,theprimersequencesareasfollows:forwardprimer:5´-ATGGGCAACTTGAAG-3´;reverseprimer:5´-GCGTCGACTTATCAGGGGCTGTTGGTG-3´.ThePCRreactionwascarriedoutat94°Cfor2min,28cyclesof94°Cfor10s,58°Cfor30sand68°Cfor240s,ina50mlvolumecontainingPMSCV-heNOSplasmid:1ml;10×buffer:5ml;2mMMgSO4:2ml;25mMdNTP(AKARABIOINC.Otsu,Shiga,Japan):5ml;KOD-pluspolymerase(ToyotoCo.,Ltd.,Osaka,Japan):1ml;primers:1mlfromeach;H2O:34.0ml.ThePCRproductwaspurifiedanddigestedbyEcoRIandSalI(NEB,Ipswich,MA),andinsertedintothePSUCMVvectorthatwasprovidedbyVirusGeneLaboratoryofEasternHepatobilearySurgicalHospitalandlinearizedbyEcoRIansSalI.ThePSUCMV-heNOSplasmidwastransformedintoE.petentcellsandpurifiedbyPlasmidMiniKit(QiagenChina,Shanghai).uracyofPSUCMV-heNOSplasmidwasverifiedbyenzymesdigestionandsequencing.ThecorrectPSUCMV-heNOSandadenoviruspackagingplasmidpBHGE3(providedbyVirusGeneLaboratoryofEasternHepatobilearySurgicalHospital)weretransfectedinto293cellsusingLipofectamine2000(Invitrogen,GrandIsland,NY).TheAd.CMV-heNOSviruswasamplifiedandpurified.Meanwhile,thevectorAd.CMVadenoviruswasgeneratedinthesameway.ResultsConstructionofpSUCMV-heNOSplasmidThefulllengthcDNAof 内容来自淘豆网www.taodocs.com转载请标明出处.
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