MakeyourtargetspecificgRNAlentivectorswithdesiredMarkers!
pLenti-gRNA-U6/H1- CloningKitisdesignedfor cloningofdouble-strandDNAencodingyourdesired gRNAsequenceforCRISPRgenomicgeneediting.Oncetranscribed,thetargetspecificgRNAwillguildCAS9enzymetocutthegenomicDNAatdesiredloci.
TomakeyourtargetspecificgRNAlentivector,twosyntheticgRNAoligonucleotidesare firstannealedtoformthedoublestandDNAduplexwhichis thenclonedintotheprovided,linearpLenti-gRNA-U6 /H1lentivectorviaT4enzymeligation.
ProductFeatures:
1)Flexibility toselectdesiredpromoter: H1orU6;
2)Flexibilitytochoose desired selectionmarker:antibioticorantibiotic-fluorescentfusiondualmarker;
3)Easyandhighefficiencycloningreaction:withprovidedpre-cut,readytouse,lineargRNAlentivector,youdoNOTneedthetedious,yetoftentroublesomelentivectorbackbonepreparation.TheCloningpositiveratecloseto100%.
4)Everythingisincludedinthiskit,enoughforgenerating10gRNAlentivectors;
5)OptionallyusedastetracyclineinducIBLe gRNAtranscription(forH1promoteronly);
ThisproductisthelentivectorversionhavingthegRNAdrivenbyaconstitutivehumanU6 promoter witha Puromycin antibiotic selectionmarkerunderRsvpromoter.PleaseseeProductManualfordetails.
Kitcontents:
pLenti-U6-(Puro)linearvector: 10ul(10rxn,1ul/rxn)
10xgRNAoligoannealingsolution: 25ul
10Xligationbuffer: 20ul
T4ligaseenzyme: 10ul(10rxn,1ul/rxn)
Cloningcontrolinsert:annealedNeg-gRNAduplex: 10 ul(10rxn,1ul/rxn)
Sequencingprimer: 10ul(10xn,1ul/rxn)DH5achemicalcompetentcells: 10vials(10xn,1vial/rxn)
ThegeneratedgRNAlentivectorcanbeusedfortransfectionorelectroporationbasedCRISPRediting,orusedforgRNAlentivirusproductionandthenusethegRNAlentivirusfor CRISPRediting.
Cat#: gRNA-U6-Puro
ebiomall.com
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量水用精细般瓶装乙醚即
采用6孔板接毒后，铺上低溶解度琼脂糖，第二天就出来了这些病变，正常细胞没有出现这种现象。
按说空斑应该出现多个，但我的六孔板中一个空最多也就出现了2个空斑，也不像文献报道的那样规则，而且很大！
由于没有做梯度稀释，会不会是接毒量过大造成的啊？请大侠指点，万分感谢！
