QIAGEN试剂盒提取RNA说明书中文翻译

1:细菌的溶菌酶裂开2:细菌的溶菌酶裂开和机械裂开3:细菌的机械裂开4:K的消化5:K的消化及机械裂开6:固体培育基细菌的裂解7:RNeasyMiniKitRNA。8:RNeasyMidiKitRNA。章节说明1-6是针对制备细菌溶7-8是针对如何从细菌溶解物中提取全RNA1-6中选择一种操作，7-8中任选其一。制备细菌溶解物的各个方案都介绍了裂开细菌时如何固定RNA长阶段和培育基的成分。细菌必需完全裂开以确保RNA的有效提取。制备细菌溶解物的各个方案都包含了不止一步。■酶消化：细菌细胞壁可以被溶菌酶进展消化〔例如溶菌酶、溶葡球菌酶。我们认为溶菌酶的作用对于全部的革兰氏阳性、阴性细菌均是有效的。■蛋白酶KK消化以提高RNAKK经常RNARNA，蛋白酶K可以有效提RNA产率。多数的细菌来说都是适用的RNA产率。尽管本手册中的机械裂开方案均为使用组织研磨机，但承受其他的裂开方法是完全可行的。考虑到细菌种类的多样性以及培育条件的多样性，细菌溶解物的制备方案〔方案1-6〕必需慎重选择。在第1011页〔表格一〕则供给了这些方案概况以便于参考选择。方案一：细菌的酶消化兰氏阳性细菌方案二：细菌的酶消化以及机械裂开该方案涉及到在机械裂开过程中使用酶进展消化根本培育基上的革兰氏阴性细菌和易于裂开的革兰氏阳性细菌。方案三：细菌的机械裂开RNA产率。方案四：细菌的酶消化和蛋白酶K处理该方案主要是溶菌酶与蛋白酶K革兰氏阴性细菌和生长在根本或简单培育基上的革兰氏阳性细菌。方案五：细菌的酶消化、蛋白酶K消化和机械裂开K用于生长在根本或者简单培育基上的难以裂开的革兰氏阳性细菌。方案六：固体培育基细菌的裂开该方案适合生长在固体培育基上的细菌白酶K或者使用机械裂开。RNeasyMiniKitRNARNeasyMiniKit100µgRNA1-6中的其中一个进展制备。RNeasyMidiKitRNARNeasyMidiKit1mgRNA。方案所需使用的细1-6中的其中一个进展制备。留意：对于某些种类的细菌或者培育条件而言，使用苯酚-异硫氰酸胍根底裂解缓冲液可以RNA的产率。该手册的附录局部含有描述通过与RNeasyLipidTissueMiniKit〔包括了苯酚-异硫氰酸胍根底裂解缓冲液〕结合使用的RNAprotectBacteriaReagent来固定并提取细菌RNA。附录C〔第41页〕是为大多种类细菌而制备的方案，介绍了溶菌酶和自行选择的蛋白酶KQIAzolRNA提取。附录D〔44页〕是为某些特定革兰氏阳QIAzolK消化、机械RNA提取。试验人员需自备的仪器和试剂因试验常接触化学药剂MSDSs，可通过供货商购置。方案须知RNase枪头■恰当型号的离心管和微型离心机或者适当转子的离心机■一次性手套■涡旋器■震荡孵育箱涉及酶解的方案〔第十一页，表一〕■胞壁质酶或者适宜的溶解酶TETrisEDTAK消化的方案〔第十一页，表一〕QIAGEN蛋白酶K涉及机械裂开的方案〔第十一页，表一〕■组织裂开器■玻璃微珠RNeasyKits的方案14.3Mb-巯基乙醇RNeasyMiniKit,RNeasyMidiKit,RNeasyProtectBacteriaMiniKit,或者RNeasyProtectBacteriaMidiKit■乙醇(96–100%)、乙醇(80%)或者乙醇(70%)■建议：RNase-FreeDNaseSet重要事项最正确的培育条件为确保基因表达的准确性以及可重复性，以下几点需试验人员留意有更少的可变因素。■收集细胞的时间：细胞应当在对数中期进展收集。在这一阶段，培育条件处于稳定状态。RNA也处于极高的水平。另外，当细菌RNAprotectBacteriaReagent固RNA的过程中降低渗透速率及效果。RNA产率受到细菌细胞世代时间很大影响，我们因此建议使用颖培育基。正确选择起始材料的使用量RNeasyKitsRNA有两个因素需要考虑：RNeasyRNA结合力：RNeasyMinispincolumn100µg，RNeasyMidispincolumn1mg。RNAprotectBacteriaReagent在细胞培育过程中的作用效果以及可以起到有效溶菌作用的RLTRNeasyMini7.5x108RNeasyMidi5x108–7.5x109细胞数目。LB培育基中生长的大肠杆菌为参考。由于不同种类的细菌会表现出Theselimitingfactorsareillustratedinthefollowing2examples,whichshowthecalculationoftheamountofE.colitoapplytoanRNeasyMinispincolumn:相关的因素将在以下的两个例子中阐述,主要是介绍了使用大肠杆菌E.coligrownin RNAyieldapproximately40µgper7.5x108cells.Uptominimalmedium7.5x108cellscanbeused(useofhighernumbersofcellsresultsininefficientlysisandreducedyield).E.coligrownin RNAyieldapproximately120µgper7.5x108cells.UptoLBmedium6x108cellscanbeused(100µgRNAisthemaximumbindingcapacityoftheRNeasyMinispincolumn).Table2showsthetypicalRNAyieldsfrombacterialcellsgrownindifferentculturemedia.Note:IftheRNAbindingcapacityoftheRNeasyspincolumnisexceeded,orifcelllysisisincompleteduetotheuseofexcessstartingmaterial,theyieldandpurityofthepurifiedRNAwillbesignificantlyreduced.BacterialspeciesCulturemediumNo.cellsRNAyield(µg)‡E.coliMinimalmedium5x10825E.coliLB5x10870B.subtilisMinimalmedium1x1088B.subtilisLB1x10815Table2.TypicalYieldsofTotalRNAfromTwoBacterialSpeciesGrowninDifferentCultureMedia**BacterialcellsdisruptedaccordingtoProtocol1.†Werecommendminimalmediaforgrowingbacteria.‡Yieldscanvaryduetofactorssuchasgenerationtimeandgrowthconditionsused.Inaddition,followingtheprotocolsformechanicaldisruptionofcells(Protocols2,3,and5)mayincreaseyields.SincetheRNeasyprocedureenrichesformRNAandotherRNAs>200nucleotides,thetotalRNAyielddoesnotincludequantitativeamountsof5SRNA,tRNA,andotherlow-molecular-weightRNAs,whichmakeup%flrIfyourstartingmaterialisneitherE.colinorB.subtilisandyoudonotknowitsRNAcontent,werecommendusingnomorethan2x108cellsperRNeasyMinispincolumnor2x109cellsperRNeasyMidispincolumninthefirstpurificationprocedure.DependingonRNAyieldandpurity,itmaybepossibletoincreasethenumberofcellsinsubsequentprocedures.TooptimizeRNAyields,werecommendperformingpilotexperimentsinwhichRNAispurifiedfromdifferentamountsofcells.QuantifyingbacterialcellsBacterialgrowthisusuallymeasuredusingaspectrophotometer.However,itisverydifficulttogivereliablerecommendationsfortherelationshipbetweenODvaluesandcellnumbersinbacterialcultures.ODreadingsareinfluencedbyfactorssuchasbacterialspeciesandphysiology,sinceODreadingsmeasurelightscatteringratherthanabsorption.Measurementsoflightscatteringdependonthedistancebetweenthesampleandthedetector,andreadingsfromdifferenttypesofspectrophotometerthereforevary.Furthermore,differentspeciesshowdifferentODvaluesatcertainwavelengths(e.g.,600nmor436nm).Bacterialphysiologycanbeinfluencedbyvariousfactors(e.g.,culturemedia,temperature,andshakerspeed).WethereforerecommendcalibratingyourspectrophotometerbycomparingODreadingsatappropriatewavelengthswithviablecelldensitiesdeterminedbyplatingexperiments.*ODreadingsshouldbebetween0.05and0.3toensurereliability.SampleswithODreadingsabove0.3shouldbedilutedsothattheODreadingsfallwithinthisrange;thedilutionfactorsareusedwhencalculatingthenumberofcellsperml.Thefollowingcalculationmaybehelpfulasaroughguide.An E.colicultureof1x109cells/mlisdiluted1:4,andgivesOD600readingsof0.25withaBeckmanDU®-7400spectrophotometeror0.125withaBeckmanDU-40spectrophotometer.ThesecorrespondtocalculatedODreadingsof1.0or0.5,respectively,for1x109cells/ml.HandlingandstoringstartingmaterialRNAprotectBacteriaReagentisaddedtobacterialculturestoimmediatelystabilizeRNA.AfterRNAstabilization,b