LiCl沉淀RNA的方法

大致分为：水相LiCl沉淀法；LiCl+乙醇法一、水相LiCl沉淀法：原理：LithiumChloride(LiCl)precipitationisaconvenientandeffectivewaytoremoveunincorporatednucleotidesandmostproteins.Lithiumchlorideprecipitation,however,doesnotprecipitatetransferRNAandmaynotefficientlyprecipitateRNAssmallerthan300nucleotides.Also,theconcentrationofRNAshouldbeatleast0.1μg/μLtoassureefficientprecipitation.特点：1、要求RNA浓度100ng/μL,才能做到有效的沉淀;400ng/μL,效果最好(对于高效的体外转录体系以及除去高RNA含量样品中的RNA污染适合)2、对于小片断(300bp)的沉淀效率较低（适用于RPA的探针纯化）。试剂：RNAPrecipitationSolution：7.5MLithiumChloride,50mMEDTA,pH8.0；方法：PrecipitationofRNAfromSolutionataConcentration≥400ng/μL1.BringthefinalconcentrationofLiClintheRNAsolutionto2.5M.2.Chillthesolutionat–20°Cfor30min.3.Centrifugeattopspeedinamicrocentrifugefor15min.4.Discardthesupernatant,andwashthepelletwithice-cold70%EtOHtoremoveresidualsalt.5.Resuspendinnuclease-freewateror10mMTris,1mMEDTA.（DEPC水）二、LiCl/ethanol沉淀法原理：即为普通的盐离子/醇法，降低核酸分子间的同电相斥，导致沉淀。特点：1、高效沉淀，无明显的片断选择性，因而对500bp的探针较为适合；2、较低浓度的RNA沉淀效率要略高于水相沉淀法；3、对RNA的沉淀特异性略低，因而沉淀产物混杂有蛋白和DNA，因而在探针制备试验中，应在沉淀前进行DNaseI处理。试剂4MLiClEthanol方法：1、invitrotranscription（20μL）；2、(Optional)Add1µLofDNasetothereactionandincubatefor15minat37°CtodigestthetemplateDNA.Add1µLof0.5MEDTAtostopthereaction.3、PrecipitatetheRNAprobebyadding2.5µLof4MLiCl,（5M加2µL，7.5M1.5µL）and100µLof100%ethanol.Optionally,add1µLof50mg/mLtRNA.4、Incubatethereactionforatleast1hat-80°C.（Centrifugeattopspeedinamicrocentrifugefor15min.Discardthesupernatant,andwashthepelletwithice-cold70%EtOHtoremoveresidualsaltRemovetheethanol.5、ResuspendtheRNApelletin50µLofRNase-freeH2O(or25µLiftheprobewassynthesizedusinghalf-volumes).Storeprobeat-80°C.Donotallowthepellettodrycompletelyafterprecipitating,oritwillbecomedifficulttoresuspend.Probesstoredat-80°Carestableforyears.