QIAGEN試劑盒提取RNA說(shuō)明書(shū)中文翻譯

1:細(xì)菌的溶菌酶裂開(kāi)2:細(xì)菌的溶菌酶裂開(kāi)和機(jī)械裂開(kāi)3:細(xì)菌的機(jī)械裂開(kāi)4:K的消化5:K的消化及機(jī)械裂開(kāi)6:固體培育基細(xì)菌的裂解7:RNeasyMiniKitRNA。8:RNeasyMidiKitRNA。章節(jié)說(shuō)明1-6是針對(duì)制備細(xì)菌溶7-8是針對(duì)如何從細(xì)菌溶解物中提取全RNA1-6中選擇一種操作，7-8中任選其一。制備細(xì)菌溶解物的各個(gè)方案都介紹了裂開(kāi)細(xì)菌時(shí)如何固定RNA長(zhǎng)階段和培育基的成分。細(xì)菌必需完全裂開(kāi)以確保RNA的有效提取。制備細(xì)菌溶解物的各個(gè)方案都包含了不止一步?！雒赶杭?xì)菌細(xì)胞壁可以被溶菌酶進(jìn)展消化〔例如溶菌酶、溶葡球菌酶。我們認(rèn)為溶菌酶的作用對(duì)于全部的革蘭氏陽(yáng)性、陰性細(xì)菌均是有效的?！龅鞍酌窴K消化以提高RNAKK經(jīng)常RNARNA，蛋白酶K可以有效提RNA產(chǎn)率。多數(shù)的細(xì)菌來(lái)說(shuō)都是適用的RNA產(chǎn)率。盡管本手冊(cè)中的機(jī)械裂開(kāi)方案均為使用組織研磨機(jī)，但承受其他的裂開(kāi)方法是完全可行的。考慮到細(xì)菌種類的多樣性以及培育條件的多樣性，細(xì)菌溶解物的制備方案〔方案1-6〕必需慎重選擇。在第1011頁(yè)〔表格一〕則供給了這些方案概況以便于參考選擇。方案一：細(xì)菌的酶消化蘭氏陽(yáng)性細(xì)菌方案二：細(xì)菌的酶消化以及機(jī)械裂開(kāi)該方案涉及到在機(jī)械裂開(kāi)過(guò)程中使用酶進(jìn)展消化根本培育基上的革蘭氏陰性細(xì)菌和易于裂開(kāi)的革蘭氏陽(yáng)性細(xì)菌。方案三：細(xì)菌的機(jī)械裂開(kāi)RNA產(chǎn)率。方案四：細(xì)菌的酶消化和蛋白酶K處理該方案主要是溶菌酶與蛋白酶K革蘭氏陰性細(xì)菌和生長(zhǎng)在根本或簡(jiǎn)單培育基上的革蘭氏陽(yáng)性細(xì)菌。方案五：細(xì)菌的酶消化、蛋白酶K消化和機(jī)械裂開(kāi)K用于生長(zhǎng)在根本或者簡(jiǎn)單培育基上的難以裂開(kāi)的革蘭氏陽(yáng)性細(xì)菌。方案六：固體培育基細(xì)菌的裂開(kāi)該方案適合生長(zhǎng)在固體培育基上的細(xì)菌白酶K或者使用機(jī)械裂開(kāi)。RNeasyMiniKitRNARNeasyMiniKit100μgRNA1-6中的其中一個(gè)進(jìn)展制備。RNeasyMidiKitRNARNeasyMidiKit1mgRNA。方案所需使用的細(xì)1-6中的其中一個(gè)進(jìn)展制備。留意：對(duì)于某些種類的細(xì)菌或者培育條件而言，使用苯酚-異硫氰酸胍根底裂解緩沖液可以RNA的產(chǎn)率。該手冊(cè)的附錄局部含有描述通過(guò)與RNeasyLipidTissueMiniKit〔包括了苯酚-異硫氰酸胍根底裂解緩沖液〕結(jié)合使用的RNAprotectBacteriaReagent來(lái)固定并提取細(xì)菌RNA。附錄C〔第41頁(yè)〕是為大多種類細(xì)菌而制備的方案，介紹了溶菌酶和自行選擇的蛋白酶KQIAzolRNA提取。附錄D〔44頁(yè)〕是為某些特定革蘭氏陽(yáng)QIAzolK消化、機(jī)械RNA提取。試驗(yàn)人員需自備的儀器和試劑因試驗(yàn)常接觸化學(xué)藥劑MSDSs，可通過(guò)供貨商購(gòu)置。方案須知RNase槍頭■恰當(dāng)型號(hào)的離心管和微型離心機(jī)或者適當(dāng)轉(zhuǎn)子的離心機(jī)■一次性手套■渦旋器■震蕩孵育箱涉及酶解的方案〔第十一頁(yè)，表一〕■胞壁質(zhì)酶或者適宜的溶解酶TETrisEDTAK消化的方案〔第十一頁(yè)，表一〕QIAGEN蛋白酶K涉及機(jī)械裂開(kāi)的方案〔第十一頁(yè)，表一〕■組織裂開(kāi)器■玻璃微珠RNeasyKits的方案14.3Mb-巰基乙醇RNeasyMiniKit,RNeasyMidiKit,RNeasyProtectBacteriaMiniKit,或者RNeasyProtectBacteriaMidiKit■乙醇(96–100%)、乙醇(80%)或者乙醇(70%)■建議：RNase-FreeDNaseSet重要事項(xiàng)最正確的培育條件為確保基因表達(dá)的準(zhǔn)確性以及可重復(fù)性，以下幾點(diǎn)需試驗(yàn)人員留意有更少的可變因素?！鍪占?xì)胞的時(shí)間：細(xì)胞應(yīng)當(dāng)在對(duì)數(shù)中期進(jìn)展收集。在這一階段，培育條件處于穩(wěn)定狀態(tài)。RNA也處于極高的水平。另外，當(dāng)細(xì)菌RNAprotectBacteriaReagent固RNA的過(guò)程中降低滲透速率及效果。RNA產(chǎn)率受到細(xì)菌細(xì)胞世代時(shí)間很大影響，我們因此建議使用穎培育基。正確選擇起始材料的使用量RNeasyKitsRNA有兩個(gè)因素需要考慮：RNeasyRNA結(jié)合力：RNeasyMinispincolumn100μg，RNeasyMidispincolumn1mg。RNAprotectBacteriaReagent在細(xì)胞培育過(guò)程中的作用效果以及可以起到有效溶菌作用的RLTRNeasyMini7.5x108RNeasyMidi5x108–7.5x109細(xì)胞數(shù)目。LB培育基中生長(zhǎng)的大腸桿菌為參考。由于不同種類的細(xì)菌會(huì)表現(xiàn)出Theselimitingfactorsareillustratedinthefollowing2examples,whichshowthecalculationoftheamountofE.colitoapplytoanRNeasyMinispincolumn:相關(guān)的因素將在以下的兩個(gè)例子中闡述,主要是介紹了使用大腸桿菌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