TAP培养基配方

TAP-Medium(Tris-Acetate-Phosphate)Lit.:Andersen,R.A.(ed.)(2005):Algalculturingtechniques,578pp,ElsevierAcademicPress,London.Gorman,D.S.&Levine,R.P.(1965):Cytochromefandplastocyanin:theirsequenceinthephotosyntheticelectrontransportchainofChlamydomonasreinhardii.-Proc.Natl.Acad.Sci.U.S.A.54:1665-1669.Harris,E.H.(1989):TheChlamydomonassourcebook:acomprehensiveguidetobiologyandlaboratoryuse.AcademicPress,SanDiego,780pp.Sueoka,N.(1960):MitoticreplicationofdeoxyribonucleicacidinChlamydomonasreinhardi.-Proc.Natl.Acad.Sci.U.S.A.46(1):83-91.TAP-MediumcanbeusedforthosealgaewhichuseNH4+insteadofNO3-asanitrogensource.Suchalgaelackthenitratereductasewhichwouldenablethemtoreducenitratevianitritetoammonium.For1000mLfinalculturemediumaddthefollowingquantities(Volume)ofstocksolutions(SL)preparedatthegivenconcentrationsto850mLdd-H2O.Addonecomponentaftertheotheruntileachonehascompletelymixedandfinallyfillupto1000mL.Allstocksolutionscanbestoredunsterilisedat4°C.StockSolution(SL)VolumeComponentConcentrationinSLConc.infinalMediumTrisbase2.42gH2NC(CH2OH)3Tris(hydroxymethyl)-aminomethan2.0010-2MTAP-salts(Beijerincksalts)25mLNH4ClMgSO47H2OCaCl22H2O15gL-14gL-12gL-17.0010-3M8.3010-4M4.5010-4MPhophatesolution1mLK2HPO4KH2PO428.8g100mL-114.4g100mL-11.6510-3M1.0510-3MTraceelementssolution(Hutnertraceelements)1mLNa2EDTA2H2OZnSO47H2OH3BO3MnCl24H2OFeSO47H2OCoCl26H2OCuSO45H2O(NH4)6Mo7O244H2O5.00g100mL-12.20g100mL-11.14g100mL-10.50g100mL-10.50g100mL-10.16g100mL-10.16g100mL-10.11g100mL-11.3410-4M1.3610-4M1.8410-4M4.0010-5M3.2910-5M1.2310-5M1.0010-5M9.2810-7MAceticacid,conc.1mLCH3COOHFirstdissolveNa2EDTA2H2Oin100mLdd-H2Obyheatingto60-80°C,thenadjustpHwithKOHto5.0.AddalltraceelementsseparatelyandcheckthepHvalueconstantly.ThepHvalueshouldnotincreaseabove6.8,otherwiseMnSO4mayprecipitate.Letthesolutionstandat4°C;whenthecolourchangesfromorangetoredafterapprox.2weeks,filterit,splitit,andstoreat-20°Cinteflonorpolycarbonatecontainers(donotuseglasscontainersfortraceelementsasthesetendtoadsorbtotheglasssurface).AfteradditionofaceticacidthepHshouldrangeatabout7.0.AdjustmediumtofinalpHof6.0orasdesiredwithaceticacidandautoclaveat121°Cfor20min.Forstockculturesonagarslantsadd1.0-1.3%Agar(e.g.purifiedhighstrength,1000gcm-²)topreparedmediumbeforeautoclaving.CCCryo4/2012