Molecular cloning chapter 07.docx

Molecular cloning chapter 07.docx

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Molecularcloningchapter07

Chapter7Extraction,Purification,andAnalysisofmRNAfromEukaryoticCells

Protocol1:

PurificationofRNAfromCellsandTissuesbyAcidPhenol-GuanidiniumThiocyanate-ChloroformExtraction

Inthissingle-steptechnique,cellsarehomogenizedinguanidniumthiocyanateandtheRNAispurifiedfromthelysatebyextractionwithphenol:

chloroformatreducedpH.Manysamplescanbeprocessedsimultaneouslyandspeedily.TheyieldoftotalRNAdependsonthetissueorcellsourceandisgenerallyintherangeof4-7µg/mlstartingtissueor5-10µg/106cells.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol2:

ASingle-stepMethodfortheSimultaneousPreparationofDNA,RNA,andProteinfromCellsandTissues

Thisprotocol,avariationofthemethoddescribedinChapter7,Protocol1,involveslysisofcellsinamonophasicsolutionofguanidineisothiocyanateandphenol.Additionofchloroformgeneratesasecond（organic）phaseintowhichDNAandproteinsareextracted,leavingRNAintheaqueoussupernatant.TheyieldoftotalRNAdependsonthetissueorcellsource,butitisgenerallyintherangeof4-7µg/mgstartingtissueor5-10µg/106cells.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol3:

SelectionofPoly（A）+RNAbyOligo（dT）-CelluloseChromatography

Chromatographyonoligo（dT）columnsisthepreferredmethodforlarge-scalepurification（>25µg）ofpoly（A）+RNAextractedfrommammaliancells.Typically,between1%and10%oftheRNAappliedtotheoligo（dT）columnisrecoveredaspoly（A）+RNA.Becausethemethodcanbefrustratinglyslow,itisnotrecommendedforpurificationofpoly（A）+RNAfrommultiplesamples.Forthispurpose,batchelution（Chapter7,Protocol4）isthebetterchoice.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol4:

SelectionofPoly（A）+RNAbyBatchChromatography

WhenmanyRNAsamplesaretobeprocessedorwhenworkingwithsmallamounts（<50µg）oftotalmammalianRNA,thetechniqueofchoiceisbatchchromatographyonoligo（dT）-cellulose.ThemethoddescribedinthisprotocolusesacombinationoftemperatureandionicstrengthtomaximizebindingandrecoveryofpolyadenylatedRNA.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol5:

SeparationofRNAAccordingtoSize:

ElectrophoresisofGlyoxylatedRNAthroughAgaroseGels

SeparationofRNAsaccordingtosizeisthefirststageinnorthernblottingandhybridization.ThemethoddescribedinthisprotocolusesglyoxaltodenaturetheRNA,ethidiumbromidetostainit,andagarosegelelectrophoresistoseparatetheresultingglyoxal-RNA-ethidiumadducts.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol6:

SeparationofRNAAccordingtoSize:

ElectrophoresisofRNAthroughAgaroseGelsContainingFormaldehyde

SeparationofRNAsaccordingtosizeisthefirststageinnorthernblottingandhybridization.ThemethoddescribedinthisprotocolusesformaldehydetodenaturetheRNA,ethidiumbromidetostainit,andelectrophoresisthroughagarosegelscontaining2.2Mformamidetoseparatetheresultingformaldehyde-RNA-ethidiumadducts.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol7:

TransferandFixationofDenaturedRNAtoMembranes

ThisprotocoldescribesthetransferofRNAfromagarosegelstoneutralorpositivelychargednylonmembranes,usingupwardcapillaryflowofneutraloralkalinebuffers.RNAbecomescovalentlyfixedtopositivelychargednylonmembranesduringtransferinalkalinebuffers.However,treatmentbyUVirradiationorheatingisrequiredtofixRNAtoneutralmembranes.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol8:

NorthernHybridization

Thisprotocoldescribeshowtocarryoutnorthernhybridizationathighstringencyinphosphate-SDS-buffers.Althoughawidevarietyofformatsareavailable,hybridizationisusuallyperformedinheat-sealablebags,rollerbottles,orplasticboxes,asdescribedhere.

IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol9:

DotandSlotHybridizationofPurifiedRNA

DotblottingofRNAisbestcarriedoutusingpurifiedpreparationsofRNAthataredenaturedwithglyoxalorformaldehydeimmediatelybeforeloadingontoanylonmembranethroughavacuummanifold.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol10:

MappingRNAwithNucleaseS1

PreparationsofRNAcontaininganmRNAofinterestarehybridizedtoacomplementarysingle-strandedDNAprobe.Attheendofthereaction,nucleaseS1isusedtodegradeunhybridizedregionsoftheprobe,andthesurvivingDNA-RNAhybridsarethenseparatedbygelelectrophoresisandvisualizedbyeitherautoradiographyorSouthernhybridization.ThemethodcanbeusedtoquantitateRNAs,tomapthepositionsofintrons,andtoidentifythelocationsof5´and3´endsofmRNAsonclonedDNAtemplates.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol11:

RibonucleaseProtection:

MappingRNAwithRibonucleaseandRadiolabeledRNAProbes

PreparationsofRNAcontaininganmRNAofinterestarehybridizedtoaradiolabeledsingle-strandedRNAprobe.Attheendofthereaction,amixtureofRNaseAandRNaseT1isusedtodegradeunhybridizedregionsoftheprobe,andthesurvivingmoleculesarethenseparatedbydenaturinggelelectrophoresisandvisualizedbyautoradiography.ThemethodcanbeusedtoquantitateRNAs,tomapthepositionsofintrons,andtoidentifythelocationsof5´and3´endsofmRNAsonclonedDNAtemplates.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Protocol12:

AnalysisofRNAbyPrimerExtension

Primerextensionisusedchieflytomapthe5´terminiofmRNAs.ApreparationofpolyadenylatedmRNAisfirsthybridizedwithanexcessofasingle-strandedoligodeoxynucleotideprimer,whichiscomplementarytothetargetRNAandradiolabeledatits5´terminus.Reversetranscriptaseisthenusedtoextendthe3´endoftheprimer.ThesizeoftheresultingcDNA,measuredbydenaturingpolyacrylamidegelelectrophoresis,isequaltothedistancebetweenthe5´endoftheprimingoligonucleotideandthe5´terminusofthetargetmRNA.

IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

Chapter7,Protocol1

PurificationofRNAfromCellsandTissuesbyAcidPhenol-GuanidiniumThiocyanate-ChloroformExtraction

Inthissingle-steptechnique,cellsarehomogenizedinguanidniumthiocyanateandtheRNAispurifiedfromthelysatebyextractionwithphenol:

chloroformatreducedpH.Manysamplescanbeprocessedsimultaneouslyandspeedily.TheyieldoftotalRNAdependsonthetissueorcellsourceandisgenerallyintherangeof4-7µg/mlstartingtissueor5-10µg/106cells.IMPORTANT:

PrepareallreagentsusedinthisprotocolwithDEPC-treatedH2O.

CAUTION

RECIPE

MATERIALS

BuffersandSolutions

Chloroform:

isoamylalcohol（49:

1,v/v）

Ethanol

Formamide（Optional）

DeionizedformamideisusedforthestorageofRNA.

Isopropanol

Liquidnitrogen

Phenol

PBS

Requiredforcellsgrowninsuspensionandmonolayersonly.

Sodiumacetate（2M,pH4.0）

SolutionD（denaturingsolution）

CellsandTissues

Mammaliancells

Mammaliantissuesamples

METHOD

1.PreparecellsortissuesamplesforisolationofRNAasappropriateforthematerialunderstudy.ThetablebelowdescribestheamountsofSolutionDrequiredforeachtypeofsample.

AmountofSolutionDRequiredtoExtractRNAfromCellsandTissues

AmountofTissue

orCells

Amountof

SolutionD

100mgoftissue

3ml

T-75flaskofcells

3ml

60-mmplateofcells

1ml

90-mmplateofcells

2ml

2.

Fortissues

a.

Isolatethedesiredtissuesbydissectionandplacethemimmediatelyinliquidnitrogen.

b.

Transferapprox.100mgofthefrozentissuetoamortarcontainingliquidnitrogenandpulverizethetissueusingapestle.Thetissuecanbekeptfrozenduringpulverizationbytheadditionofliquidnitrogen.

c.

Transferthepowderedtissuetoapolypropylenesnap-captubecontaining3mlofSolutionD.

d.

Homogenizethetissuefor15-30secondsatroomtemperaturewithapolytronhomogenizer.

3.Formammaliancellsgrowninsuspension

a.

Harvestthecellsbycentrifugationat200-1900g（1000-3000rpminaSorvallRT600usingtheH1000rotor）for5-10minutesatroomtemperatureinabenchtopcentrifuge.

b.

Removethemediumbyaspirationandresuspendthecellpelletsin1-2mlofsterileice-coldPBS.

c.

Harvestthecellsbycentrifugation,removethePBScompletelybyaspiration,andadd2mlofSolutionDper106cells.

d.

Homogenizethecellswithapolytronhomogenizerfor15-30secondsatroomtemperature.

4.Formammaliancellsgrowninmonolayers

a.

Removethemediumandrinsethecellsoncewith5-10mlofsterileice-coldPBS.

b.

RemovePBSandlysethecellsin2mlofSolutionDper90-mmculturedish（1mlper60mmdish）.

c.

Transferthecelllysatestoapolypropylenesnap-captube.

d.

Homogenizethelysateswithapolytronhomogenizerfor15-30secondsatroomtemperature.

5.Transferthehomogenatetoafreshpolypropylenetubeandsequentiallyadd0.1mlof2Msodiumacetate（pH4.0）,1mlofphenol,and0.2mlofchloroform-isoamylalcoholpermilliliterofSolutionD.Afteradditionofeachreagent,capthetubeandmixthecontentsthoroughlybyinversion.

6.Vortexthehomogenatevigorouslyfor10seconds.Incubatethetubefor15minutesonicetopermitcomple