LiClRNAPreparation(AmbrosLab)

2)Grindtoafinepowderinamortarthatwasprecooledwithliquidnitrogen

3)TransferpowdertoaFalcontube

4)Addanequalvolumeof2XLETS(i.e.,equaltotheoriginalvol.ofworms)

5)Addanequalvolumeofphenol:chloroform(i.e,twicetheoriginalvol.ofworms)

6)Vortexandshakeuntilpowderiscompletelythawed

7)Separatephasesinaclinicalcentrifugeat3000rpm,about5-10minutes,roomtemperature

8)Extractwithphenol:chloroform3xormoreuntilinterfaceisalmostcompletelyclear

9)PrecipitatebyaddingLiCLto0.2Mand3volumesof100%ETOHandputat-20oCfor30minutesorwhateverisconvenient(Igenerallyobserveaprecipitateimmediately)

10)Collectprecipitatebycentrifugationinclinicalcentrifuge,3000rpm,10-15minutesroomtemperature

11)Rinsepelletseveraltimeswith70%ETOHandresUSPendinDEPCtreatedwaterorTE

12)ReadOD260/280onanappropriatedilution:aratioof2.0impliesafairlypurepreparationofRNA

13)ExamineonadenaturinggeltoassesstheintegrityoftheRNA

14)StoreRNAasanethanolprecipitateorat-70oC

(ThetraceamountofDNAcontaminationcanberemovedwithDNAse,ifnecessary)

2XLETS200mMLiCL20mMEDTA20mMTrispH7.82%SDS(Iaddvanadylribonucleosidecomplexesto2mMjustbeforeuse-fromSigma)

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