northern protocol

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1、RULESFORRNAWORK1.Wearglovesatalltimeincludingfillingpipettipinracks,fillingjarswithEppendorftubes,andweighingchemicalstopreparesolutions.2.Pullweighingpaperorweighingboatfrommiddleofpile.Tapchemicalsoutofbottles.Useweighingboatsorspecimencupsfoundinchemicalsinbucketsf

2、orspooningthosechemicalsoutforweighing.3.Designatespecificbottlesforeachsolution.Topreparenewbatchofsolution,justrinsebottleswithultrafiltered(UF)water.4.AllsolutionstobeautoclavedarepreparedinUFwaterandautoclavedfor40min.Solutionsnottobeautoclavedarepreparedinautocla

3、vedwater.5.Disposablesaresterilizedbybakingat1100Cfor2hr.6.Metalandglassaresterilizedbybakingat2200Covernight.21RNAExtractionfromCulturedCellsImportantTip:Tomaximizeyield,processsamplesthroughminimumnumberoftubes.Foreachexperimentalgroup,youonlyneed1syringe/needle,1sc

4、raper,and1ultracentrifugetube.I.CellLysis(inchemicalhood)1.Labelultracentrifugetubesonbottom.Addabout2mlofusedGITtotubestoremovepotentialRnasecontamination.Covertubeswithplasticwraporpapertowel.2.PourenoughGITforthecurrentpreparationina50mlconicaltube.Inhood,add2-merc

5、aptoethanoltoafinalconcentrationof2%.3.Removeculturedishesfromincubator.4.Aspirateallliquid.Keepdishinvertedtodrain.Aspirateagain.5.Add2mlGITtoeachdish.Swirltocoverentireplate.6.Scrapedishwithscraper.7.Collectcelllysatein10mlsyringewith23Gneedle.8.Addadditional1mlGITt

6、odishandscrapeagain.9.Combinelysateandrinsefromthesametreatmentgroupinthesamesyringe.10.PushlysatethroughneedleintoacornerofoneofthedishestwotimestoshearDNA.*Ifyouworkwithtissue,duringthesecondtime,takeupalso50mlof30%sarkosyl(afinalconcentrationof0.5%).11.Samplescanbe

7、storedinsyringe,inbox,at-800Cuntilreadyforfurtherprocessing.21II.Ultracentrifugation1.PourGITbackto"usedGITtube"fromultracentrifugetubes.2.Add1.8mlof5.7MCsCltoeachtube.3.OverlaysamplesfromstepI.9overCsClcarefully.Avoidmixingthetwosolutions.4.Centrifugeat36,000rpm,200C

8、for17hoursorlonger.*Loadall6bucketsatalltimeforcentrifugation!*Numberonbucketsshouldmatchnumberonrotor.21III.RNARecovery1.Re