Agarose-FormaldehydeElectrophoresis

Youwillneed:-

10xMOPSbuffer:

0.2MMOPS(morpholinopropanesulphonicacid)50mMsodiumacetate5mMEDTAThebufferisadjustedtopH7.0with1MNaOHandsterilisedbyautoclaving.

RNAdenaturingbuffer:

10ml100%deionizedformamide3.5ml40%formaldehyde1.5ml10xMOPSbufferFormamideisdeionizedbystirring100mlwithapproximately20gofAmberliteMB3(orMB1)ionexchangeresinfor15minutes.

N.B:Careshouldbetakenhandlingsolutionscontainingformaldehyde.Formaldehydeisextremelytoxic.Glovesshouldbewornwhenpreparingandhandlingsolutionscontainingformaldehyde.Electrophoresisutilisingformaldehydecontainingbuffersshouldbeperformedinawellventilatedareai.e.afumehood.

Agaroseispreparedbymeltingtherequiredamountofagaroseindistilledwater,coolingtoapproximately60°C(handhot)andadding40%formaldehydeand10xMOPStogive2.2Mformaldehydeand1xMOPS,respectively.Example:For50mlofa1%agarosegel,melt0.5gagarosein37mlH₂O,cooltohandhot,add5ml10xMOPSbufferand8.75ml40%formaldehyde.

Electrophoresisisin1xMOPS,2.2Mformaldehyde.

RNAsamplesarepreparedbyaddingupto25mgofRNAinamaximumof5ulsterileH₂O,to15ulRNAdenaturationbuffer.1ul10mg/mlethidiumbromideisaddedtoaidvisualisationofRNAafterelectrophoresis.

N.B.:CareshouldbetakingonremovalofthegelformingcombspriortoloADIngofsamplesasruptureofthewellbottomscanoccur.Agarose/formaldehydegelsareinherentlyweakerthantheequivalentpercentageagarosegels.

Immediatelypriortoloading,RNAsamplesareheatedto65°Cforapproximately10minutestodenatureanysecondarystructure,cooledonicefor2minutesand2ulofsterileloadingbufferadded.

Samplesareloadedontothegelandelectrophoresedatnomorethan5V/cm,withoccasionalbufferrecirculation,untiltheleadingbromophenolbluedyefronthasmigratedapproximatelythreequartersofthelengthofthegel.VisualisationofRNAisachievedbyirradiationwithshortwave(254nm)UVlight.TypicalMarkersofRNAqualityare18S(~1900bases)and28S(~4800bases)RNAmolecules.

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