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1、------------------------------------------作者xxxx------------------------------------------日期xxxx原生质体制备【精品文档】本氏烟草原生质体制备SchweigerRandSchwenkertS.2014.Protein-proteinInteractionsVisualizedbyBimolecularFluorescenceComplementationinTobaccoProtoplastsandLeaves.JournalofVisualizedExperiments.85
2、(51327):1-8.1ProtoplastPreparationProtoplastpreparationoftobaccoleaveswasadaptedfromKoopetal.16andslightlymodified.1.1Bufferpreparation1.PrepareF-PCNmedium:Macro-salts[KNO3(1012μg/ml),CaCl2•2H2O(440μg/ml),MgSO4•7H2O(370μg/ml),KH2PO4(170μg/ml),NH4-succinate[(20mM;preparea2Mstocksolution(s
3、uccinate(236μg/ml)andNH4Cl(106μg/ml),adjusttopH5.8todissolve)],Micro-salts[EDTA-Fe(III)xNa-salt(40μg/ml),KJ(0.75μg/ml),H3BO3(3μg/ml),MnSO4•H2O(10μg/ml),ZnSO4•7H2O(2μg/ml),Na2MoO4•7H2O(0.25μg/ml),CuSO4•5H2O(0.025μg/ml),CoCl2•6H2O(0.025μg/ml)],MES(390μg/ml),glucose(approximately80μg/ml)osm
4、olarity550mOsm,pH5.8(KOH).Storeinaliquotsat-20°C.2.PrepareF-PINmedium:AllingredientsasF-PCNbutinsteadofglucoseusesucrose(approximately110μg/ml),osmolarity550mOsm,pH5.8(KOH).Storeinaliquotsat-20°C.3.PrepareW5medium:150mMNaCl,125mMCaCl2,5mMKCl,2mMMES,osmolarity550-580mOsm,pH5.7(KOH).Storea
5、t4°C(sterilefilterthrougha0.2μmfiltertopreventbacterialgrowthduringlongerstorage).4.Preparefreshenzymesolutionforprotoplastisolation(0.1gcellulase,0.03gmacerozymin10mlF-PIN).Incubatethesolutionat55°Cfor10minandcooltoRT.Add100μlof10%BSAto10mlsolution.【精品文档】【精品文档】1.2Isolationofprotoplasts1
6、.PlaceoneinfiltratedleafintoaPetridishandaddthefreshenzymesolution.Useanewrazorbladetocuttheleafintoapproximately0.5cm2sizedpieces.Transfertheleaf-pieceswiththeenzymesolutionintoavacuum-infiltrationflaskandvacuuminfiltrateforapproximately20secuntilairbubblesemergefromtheleaves(releasevac
7、uumverycarefully).2.Shaketheflaskfor90minat40rpmindarkness.3.Releaseprotoplastsbyshakingfor1minat90rpm.Filterthesolutionthroughgauze(100μM)intoa15mlcentrifugationtube(roundbottom).4.Overlaytheprotoplastsolutionwith2mlF-PCNbufferandcentrifugefor10minat70xg(slowacceleration
