mRNA Purification
来自 : 蚂蚁淘
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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