6.PlacetheBeadTubeontheVortexAdapterandvortexatmaximumspeedfor10minutes.EmxvxOtOco
最大转速漩涡混合10min。
What’shappening:
Cellsarelysedbycombinationofchemicalagentsfromsteps1-5andthemechanicalshakingintroducedbyvortexing.Phenol:
chloroform:
Isoamylalcoholisaddedtomaximizelysingefficiencyandyield.Lysedcellcomponentsaretrappedinthesolventandproteinsaredenaturedleavingthenucleicacidinsolution.SixE2yXPq5
反应：
从1到5步的化学试剂和漩涡混合使细胞裂解，酚/氯仿/异戊醇使其最大程度裂解。
溶解的细胞和试剂混合在一起，蛋白质降解只剩下核酸在溶液中。
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TheMOBIOVortexAdapterisdesignedtobeasimplecosteffectiveplatformtofacilitatethekavU42VRUs
homogenizationandcelllysisofsamples.AnalternativetotheMOBIOVortexAdapterwouldbetoattachyourtubestoyourplatformwithtape.Notethattapecanbecomelooseandmayresultinunevenshakingandlysisefficiencyresultingininconsistentresultsorloweryields.y6v3ALoS89
7.RemovetheBeadTubefromtheVortexAdapterandcentrifugeat2500xgfor10minutesatroomtemperature.M2ub6vSTnP
What’shappening:
Centrifugationresultsinphaseseparationofthesamplemixture.Threephaseswillbevisibleaftercentrifugation.Thelowerorganicphasecontainingproteinsandcellulardebris,theinterphasecontaininghumicsandotherorganicandnon-organicmaterial,andtheupperaqueousphasecontainingtotalnucleicacid.Note:
Thethicknessoftheinterphasewilldependonthesampletype.Sampleshighinorganiccontentwillhaveathickerinterphase.0YujCfmUCw
7、室温2500g离心10min。
反应：
离心导致混合样品相分离。
离心后能观察到三相，底下的有机相包括蛋白质和细胞碎片，中间相包括腐殖质和其他有机及无机物质，上层相包括所有的核酸。
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注意：
中间相的厚度和样品类型有关，样品有机物含量越高，中间相越厚。
8.RemovetheBeadTubefromthecentrifugeandcarefullytransfertheupperaqueousphase（avoidingtheinterphaseandlowerphenollayer>toaclean15mlCollectionTube（provided>.Thethicknessoftheinterphasewillvarydependingonthetypeofsoilused.Discardthephenol:
chloroform:
isoamylalcoholinanapprovedwastereceptacle.Note:
Thebiphasiclayerwillbethickandfirminsoilshighinorganicmatterandmayneedtobepiercedtoremovethebottomphenollayerfordisposal.sQsAEJkW5T
8、小心地移取上层水相到一15ml收集管中。
注意：
有机物含量高两相层可能会较厚较坚固，可能需要移取底层的酚相来处理。
What’shappening:
Theupperaqueousphasecontainingthetotalnucleicacidsfromthesampleistransferredtoanewtube.Cellulardebris,proteins,andorganicmaterialareleftbehind.Note:
Takecarenottotransfermaterialfromthelowerphaseortheinterphase.GMsIasNXkA
反应：
包括样品总核酸上层水相转移到新收集管中。
丢弃细胞碎片，蛋白质和有机物。
注意：
不要碰到中间相和下层酚相。
9.Add1.5mlofSolutionSR3totheaqueousphaseandvortextomix.Incubateat4°Cfor10minutes.TIrRGchYzg
What’shappening:
SolutionSR3isasecondaryprecipitationsteptofurtherremoveproteinsandcellulardebris.7EqZcWLZNX
9、加1.5mlSR3到水相中，漩涡混合。
4℃静置10min。
反应：
加SR3是二次沉淀步骤，进一步去除其中的蛋白质和细胞碎片。
10.Centrifugeat2500xgfor10minutesatroomtemperature.lzq7IGf02E
10、室温2500g离心10min。
11.Transferthesupernatant,withoutdisturbingthepellet,toanew15mlCollectionTube（provided>.zvpgeqJ1hk
What’shappening:
Thesupernatantcontainingnucleicacidsaretransferredtoanew15mltube.Nonnucleicacidmaterialisleftbehind.NrpoJac3v1
11、将上清液转移到15ml收集管中<不要碰到下面的沉淀物）。
反应：
包括核酸的上清液转移到新的收集管中，剩下非核酸物质。
12.Add5mlofSolutionSR4totheCollectionTubecontainingthesupernatant,invertorvortextomix,andincubateat-20°Cfor30minutes.1nowfTG4KI
Note:
Forsampleswithhighsaltcontent,incubationinSolutionSR4atroomtemperaturewillresultinhigherRNAyields.fjnFLDa5Zo
12、加5mlSR4到收集管中，倒置混合，-20℃静置30min。
注意：
对于含盐量高的样品，室温条件下加SR4静置将得到高RNA产量。
13.Centrifugeat2500xgfor30minutesatroomtemperature.tfnNhnE6e5
13.、室温2500g离心30min。
14.Decantthesupernatantandinvertthe15mlCollectionTubeonapapertowelfor5minutes.HbmVN777sL
Note:
Dependingonsoiltype,thepelletmaybelargeand/ordarkincolor.V7l4jRB8Hs
What’shappening:
SolutionSR4is100%Isopropanol.NucleicacidisprecipitatedandtheIsopropanolisdiscarded.83lcPA59W9
14、倒出上清液，将收集管倒置在纸巾上5min。
注意：
依据土壤类型的不同，沉淀可能较大或颜色上较深。
反应：
SR4是纯异戊醇。
沉淀核酸，丢弃上清液。
15.ShakeSolutionSR5tomix.Add1mlofSolutionSR5tothe15mlCollectionTubeandresuspendthepelletcompletely.（Note:
Dependingonthesoiltype,thepelletmaybedifficulttoresuspend.Resuspensionmaybeaidedbyplacingthetubesinaheatblockorwaterbathat45°Cfor10minutes,followedbyvortexing.Repeatuntilthepelletisresuspended.>mZkklkzaaP
What’shappening:
SolutionSR5isaproprietarysaltsolutionusedtoresuspendtheprecipitatednucleicacidsfromstep14.ItisalsousedtoequilibratetheRNAcapturecolumninstep16andtowashandprepthecolumnfortheelutionofRNAinstep20below.AVktR43bpw
15、摇晃SR5使其混合，加1mlSR5到收集管中，使沉淀再完全悬浮。
注意：
土壤样品不同，沉淀有可能不易悬浮，可能需要将收集管放到45℃的水浴池中10min再悬浮,再漩涡混合，重复这样直到沉淀重悬浮。
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16.PrepareoneRNACaptureColumn（provided>foreachRNAIsolationSample:
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a.Removethecapofa15mlCollectionTube（provided>andplacetheRNACaptureColumninsidethe15mlCollectionTube.Thecolumnwillhanginthe15mlCollectionTube.gIiSpiue7A
b.Add2mlofSolutionSR5totheRNACaptureColumnandallowittogravityflowuEh0U1Yfmh
throughthecolumnandcollectinthe15mlCollectionTube.AllowSolutionSR5toIAg9qLsgBX
completelyflowthroughthecolumn（Optional:
TheCollectionTubemaybeemptiedafterSolutionSR5hascompletelyflowedthroughthecolumn.Note:
DONOTALLOWTHECOLUMNTODRYOUTPRIORTOLOADINGTHERNAISOLATIONSAMPLE.>WwghWvVhPE
16、为每个RNA样品准备一个捕集柱。
a.将捕集柱悬挂到收集管上。
b.加2mlSR5到捕集柱上，使其重力流。
允许SR5完全流过捕集柱。
注意：
在加RNA样品前不要让捕集柱流干。
17.AddtheRNAIsolationSamplefromStep15ontotheRNACaptureColumnandallowittogravityflowthroughthecolumn.Collecttheflowthroughinthe15mlCollectionTube.asfpsfpi4k
17、将15步的RNA分离样加到捕集柱中，使其流过捕集柱。
18.Washthecolumnwith1mlofSolutionSR5.Allowittogravityflowandcollecttheflowthroughinthe15mlCollectionTube.ooeyYZTjj1
What’shappening:
ThesampleisaddedtotheRNACaptureColumnandthenucleicacidsareboundtothecolumnmatrix.TheCaptureColumnisthenwashedwithasecondvolumeofSolutionSR5toensureunboundcontaminantsareremovedfromthesampleandcolumnpriortotheelutionofRNA.BkeGuInkxI
18、用1mlSR5洗涤捕集柱，流出液收集在收集管中。
反应：
样品加到捕集柱上，核酸结合到柱基质上。
捕集柱用SR5洗涤确保没结合的污染物被去除掉。
19.TransfertheRNACaptureColumntoanew15mlCollectionTube（provided>.ShakeSolutionSR6andthenadd1mlofSolutionSR6totheRNACaptureColumntoelutetheboundRNAintothe15mlCollectionTube.AllowSolutionSR6togravityflowintothe15mlCollectionTube.PgdO0sRlMo
What’shappening:
TheSolutionSR6RNAelutionbufferisaproprietarysaltsolutionthatallowsforthepreferentialreleaseofRNAfromtheRNACaptureColumnleavingDNA,residualdebris,andinhibitingsubstancesinthecolumn.Note:
anewkitisavailableforDNAelution.SeeDNAElutionProcedureintheHintsandTroubleshootingGuideorcontactMOBIOfordetailsattechnical@3cdXwckm15
19、将捕集柱转移到新收集管中，摇晃SR6，然后加1mlSR6到捕集柱中使其流过捕集柱，洗提RNA。
反应：
SR6RNA洗提缓冲液是专有的盐溶液，它能使RNA流出而DNA、剩下的细胞碎片和抑制剂依然留在捕集柱上。
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20.TransfertheelutedRNAtoa2.2mlCollectionTube（provided>andadd1mlofSolutionSR4.Invertatleastoncetomixandincubateat-20°Cfor10minutes.v4bdyGious
20、将洗提的RNA转移到2.2ml的收集管中，并加1mlSR4，至少倒置混合一次，-20℃静置10min。
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21.Centrifugethe2.2mlCollectionTubeat13,000xgfor15minutesatroomtemperatureto
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