mRNAPurification

I.Prepareoligo-dTcellulose

• use40mgoligo-dTcellulose/1mgtotalRNA
• swelloligodT-celluloseinelutionbuffer
• washoligodT-cellulose4xwithelutionbuffer(30sec.fullspeedspininbetween)
• equilibrateoligo-dTcellulosewith2to3washingstepsusing1xbindingbuffer

II.mRNApurification

• bring1mgtotalRNAwithH₂Oto600µl
• incubate4minat65°C
• add600µl2xbindingbuffer
• addto40mg1xbindingbufferequilibratedoligo-dTcellulose
• incubate15minatRTonarollingincubatororvortexseveraltimesinbetween
• spinoligo-dTcellulosedown,discardsupernatant
• wash2xwith1xbindingbuffer
• wash2xwithwashbuffer
• elutewith250µlelutionbufferat37°Cfor5min
• spinandkeepsupernatant(forsecondroundofpurificationorprecipitation)
• eluteoligo-dTcelluloseagainwith250µlelutionbuffer
• spinandkeepsupernatant(forsecondroundofpurificationorprecipitation)
• combineeluateandaddH₂Oto600µl
• repeatmRNApurificationbystartingagainwith4minincubationat65°C

III.RecovermRNA

• add50µl4MNaClandprecipitatewith2vol.coldEtOH
• incubate1hat-20°C
• spin10minfullspeed,washwith70%EtOH,airdryanddissolvein20µlH₂O
• readA₂₆₀of1µl(100to250folddiluted)
• run1µg(and10µgtotalRNAascomparison)ona1.2%agaroseFormaldehydegelrecovery=1-2%ofthetotalRNA(10-20µgmRNA/2mgtotalRNA)

Buffers:[allbuffersshouldbemadewithDEPCtreated/autoclavedcomponents!]oligodTcellulose(type7,Pharmacia)2xbindingbuffer:1MNaCl;20mMTrispH7.5;2mMEDTA;0.1%SDS[50ml:12.5ml4MNaCl;1ml1MTris;200µl0.5MEDTA;500µl10%SDS]1xbindingbuffer:0.5MNaCl;10mMTrispH7.5;1mMEDTA;0.05%SDS[50ml:6.25ml4MNaCl;500µl1MTris;100µl0.5MEDTA;250µl10%SDS]washbuffer:0.2MNaCl;10mMTrispH7.5;1mMEDTA;0.05%SDS[50ml:2.5ml4MNaCl;500µl1MTris;100µl0.5MEDTA;250µl10%SDS]elutionbuffer:10mMTrispH7.5;1mMEDTA;0.05%SDS[50ml:500µl1MTris;100µl0.5MEDTA;250µl10%SDS]

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