二氧化碳培养箱的使用的注意事项
来自 : 蚂蚁淘
PhenolExtractionofrRNA(Ratliver)LEVELII
***READTHROUGHALLCAUTIONSBEFORETRYINGTHISEXPERIMENT***Materials
- Ratliver(fastedrat)
- Liquidnitrogen
- p-Amino-salicicacid
- Phenolmixture
- Homogenizerorblender6
- Refrigeratedpreparativecentrifuge
- NaCl
- 95%and70%(v/v)ethanol
Procedure
- Obtainaratwhichhasbeenfastedfor24hours(toremoveglycogenfromtheliver),decaptitate,exsanguinateandremovetheliverasrapidlyaspossIBLe.
- Weightheliver,beingcarefulnottoallowtheittodehydrate.
- Immediatelydroptheliverintoacontainerofliquidnitrogen.
CAUTION:Liquidnitrogenwillcauseseverefrostbite!
- Usingtheweightoftheliverasanindicationofthevolume(1gmofliverequivalentto1ml),add15volumesoffreshlyprepared6%para-amino-salicylate(pAS)toachilledblenderorhomogenizer.
- Addanequalvolume(equaltothepAS)ofphenolmixturetotheblenderandturnontheblenderforashortbursttomixthepASandphenol.
CAUTION:Phenolisextremelycaustic.
Phenolcausessevereskinburns,yetitisalocalanesthetic.Youwillbeunawareoftheburnatfirst,exceptfortell-talediscolorationoftheskinandblisters.Youwillbecomeawareoftheburnastheanestheticpropertieswearoff.PhenolalsoreADIlydissolvesmostcountertopsandallrubbercompounds.
CLEANUPALLSPILLSIMMEDIATELY!NOTIFYYOURINSTRUCTOROFANYSPILLSAFTERYOUHAVETHOROUGHLYRINSEDANDWASHEDAWAYANYMATERIALSINCONTACTWITHYOURSKIN.
- Stoptheblenderandaddthefrozenliver(handletheliverwithlongforceps,ortongs).Blendtheentiremixture(pAS,phenolandliver)for30secondsatfullspeed.DonotblendforlongerperiodsoryouwillsheertheRNA.
- Carefullytransferthehomogenatetoabeakerandcontinuetostirthemixturefor10minutesatroomtemperature.
- Transferthehomogenatetonalgenecentrifugetubesandcentrifugethemixtureat15,600xgat4°Cfor20minutes.
- Removethecentrifugetubesandcarefullyseparatetheupperaqueouslayerfromthelowerphenollayer.Takecarethatnoneofthewhiteinterphasematerialismixedintotheaqueouslayer.Theupperlayercanmostefficientlyberemovedbyusingalargehypodermicequippedwithalong,largebore,squaretippedneedle.Shouldsomeoftheinterphasematerialbestirredintotheaqueousphase,itwillbenecessarytorepeatstep8.
- Measurethevolumeoftheaqueouslayeranddiscardthephenollayerandinterphasematerial.
- Add3.0gramsofNaClper100ml.ofaqueousphaseandstiruntildissolved.
- Add0.5volumesofphenolmixturetotheaqueousphase,placeintoasuitableflaskandshakevigorouslyforaboutfiveminutes.Recentrifugeasinstep8above,butfor10minutes.
- Separatetheaqueousphaseandadd2.3volumesofcold95%ethanol.Allowthemixturetostandinthefreezeruntilaprecipitateforms.
- CollecttheRNAprecipitatebycentrifugation,washoncein70%ethanolandstorein70%ethanolat0-5°C.
Notes
KnowledgeoftranscriptionisbasedonourABIlitytoextract"native"orfunctionalRNAmoleculesfromcells,withsubsequentuseofthosemolecules"invitro."Oneoftheearliestmethodsforthistypeofanalysisisaphenol-detergentextractionofRNA7coupledwithseparationofthevarioussizedmoleculesofRNAwithcentrifugationinagradient.
Thisbasicprocedureremainsusefultoday,althoughtherehavebeenmyriadadditionsandalterationstotheprocedureusingahostofextractiontechniquesandseparationprocedures(suchaselectrophoresisorcolumnchromatography).
Forthepurposesofintroductiontothetechnique,thisexerciseextractsRNAfromratliverusingaphenolextractionwhichyieldspredominantlyrRNAandtRNA.ThereissomemRNApresent,butitisvariableandshouldbeconsideredasabackgroundcontaminant.ThereisalsoagoodportionofsRNAcausedbysheeringoftheRNAduringhomogenization,andbyenzymaticdigestionbyRNAaseduringtheextraction.
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