2.Transfertheground,frozenwormpowderdirectlyintoaCorningtubecontainingtheappropriatevolumeoflysisbuffer(Igenerallyuse4timesthevolumeofpackedworms).Thesolutionwillinstantlyfreezetoaslurry,butthissoonthawsinthe65degreeincubation.IncubatethesUSPensionat65degreesfor30-60minutes.Itshouldbecomereasonablyclear.

Lysisbuffer:

1ml0.5MEDTA2ml1MTris,pH7.54ml20%SDSwaterto40mladd100ul20mg/mlproteinaseK(stockinTE,storedfrozeninaliquotsat-20degrees;thestuffI"vebeenusingishighgradefromMerck-youwanthighlypurifedinordertoavoidRNasecontamination)

3.Phenolextractextensively.Iusuallybackextractthefirstinterfacewith1-2mloflysisbuffer,andcombinetheaqueousphasefromthiswiththatfromthefirstextractionforallsubsequentextractions."Extensively" = at least 4-5 extractions. There should be very little interface by the last one. Then do a couple of chloroform extractions for good measure. (Thorough extraction is a critical step in this protocol - the times I"ve gotten degradation of the RNA have been when I"ve cut corners here.)

4.AddLiCltoafinalconcentrationof2M(froma10Mstocksolution).Incubateonice2-3hours(longerisOK)orovernightat4degrees.(LiClprecipitatesRNA,leavesDNAbehind.)

5.Spindown.Avarietyofcentrifugationswillwork:SW55intheultracentrifuge,30krpmfor15min.at4degrees(thisisoverkill);JA20,8krpmfor20-30min.;Eppendorffor15-20min.;etc.

6.ResuspendtheRNApelletin1-3mlofTES(TES=10mMTris7.5,1mMEDTA,0.1-0.2%SDS).ChoosethevolumebasedonhowmuchRNAyouhave;LiClprecipitationsseemtonotworkverywellwhentheRNAconcentrationfallsbelowsomethreshold,sokeepthevolumeassmallasisreasonableforgettingyourpelletresuspended.Thepelletscanbequitedifficulttoresuspend,andsometimesIresorttoshortincubationsat65degreestospeedtheprocess,thoughthisalwaysmakesmenervousaboutlurkingRNases.

7.AddLiClto2Mandrepeattheprecipitation.

8.RepeattheLiClprecipitationathirdtime.

9.ResuspendtheRNAinTES.Phenolextract(justaninsurancemeasureagainstanystrayRNasethatmayhavewanderedinduringtheprecedingmanipulations).Ethanolprecipitate.Washthepellet2timeswith70%ethanol.DryandresuspendinthedesiredvolumeofTESorTEorwater,dependingonwhatyouwanttousetheRNAfor.Storeat-20degrees.TESisbestifallyou"regoingtodoisrunNortherns-theSDSisapartialinsuranceagainstRNase.ButasSDSisincompatIBLewithmostenzymaticrecations,ifyou"regoingtobemakingCDNA,etc.,you"llnotwanttouseTES.Ifyou"vedoneagoodjobonthepurification,yourRNAwillbestableat-20withouttheSDS.

AnnSluder