农杆菌培养与转化

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1、B.Gould2007TransformationandstorageofVIGSagrobacteria:Onceyoursilencingvectorsequencehasbeenverified,transformasampleintoelectrocompetentagrobacteria,strainGV3101(ortryEHA105).Beforetransformationverifythattheelectroporator,electrocompetentagrobacteriacells,andappropriateselectiveplatesareavailable.

2、Foraprocedureonhowtomakeandtransformelectrocompetentagrobacteriacellsrefertop.122ofWeigelandGlazebrook’s“Arabidopsis:ALaboratoryManual”.AgrobacteriumGV3101hasrifampicinresistance(at25ug/mL,chromosomalmarker)andgentamycinresistance(at50ug/mL,Tiplasmidmarker).TheTRV2andTRV1vectorsbothconferkanamycinre

3、sistanceat50ug/mL.(AgrobacteriumEHA105hasbackgroundchromosomalresistancetorifampicin(25ug/mL)andchloramphenicol(25ug/mL),withnoantibioticmarkeronitsTi-plasmid.)Forpouringnewantibioticplatesandgrowingliquidculturesusethefollowingantibioticstocksanddilutions:AntibioticPowderstorage:StocksolutionFinalc

4、oncentrationuLstock/mLmediaKanRoomtemp50mg/mLinwater50ug/mL1Gn4degC50mg/mLinwater50ug/mL1Rif-20degC25mg/mLinmethanol*25ug/mL1Storeantibioticstocksolutionsat-20degC.Makeinaliquotsanddiscardafterrepeatedfreeze-thaws.*doNOTusebluecapFalcontubesformixingmethanolsolutions(bluedyewilldissolveintosolution)

5、.PCRscreenagrocolonies(aspreviouslydonefore.coli)using156Fand156Rprimers.Pickonecolonywiththecorrectinsertsize,andgrowa3mLovernightculture.Useasterileculturetubeandshakeovernightatabout270rpmatroomtemperatureinGn50Kan50Rif25LBmedia.Makeafewglycerolstocksfromthisculturebydoingthefollowing:Þprepare60%

6、sterileglycerolinwaterbyfiltersterilization(donotautoclaveglycerolsolutions),andobtainasmallquantityofliquidnitrogen.ÞLabelthreesterilescrew-capplastictubes.Ineachtubemix0.333mLof60%glycerolsolutionand1mLofculture(ingeneralusea1:3glyceroltocultureratio)intoeachtubeandpipettethemixtureslowlyupanddown

7、tomix.ÞCloseeachtubeandplaceintoliquidnitrogentosnap-freeze.Afteraminuteorso,removethetubesandstorethemat-80degC.B.Gould2007GrowingVIGScultures:1.Createfreshstreakplatesofeachagrobacteriaculturetobeus