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Thermo Fisher Scientific Purification of RNA Transcripts User guide

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Protocol
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Purication of RNA Transcripts
• Template DNA:mayinterferewithdownstreamapplicationsoftheRNAtranscript.TemplateDNAshouldbe
removedbyDNaseIdigestiondirectlyafterthetranscriptionreaction.Add2uofDNaseI,RNase-free,mixand
incubateat37°Cfor15min,thenadd2µlof0.5MEDTA,pH8.0andincubateat65°Cfor10mintostopthe
reaction.
• Proteins and nucleotides:phenol/chloroformextractionandethanolprecipitationofRNAtranscriptsis
recommended.
1. To50µlreactionmixture,add85µlofDEPC-treatedwaterand15µlof3MSodiumAcetate.Mixthoroughly.
2. Extractwithanequalvolumeof1:1phenol/chloroformmixture,andthentwicewithanequalvolumeof
chloroform.Collecttheaqueousphaseandtransfertoanewtube.
3. PrecipitatetheRNAbyadding2volumesofethanol.Incubateat-20°Cforatleast30minandcollectthepellet
bycentrifugation.
4. Removethesupernatantandrinsethepelletwith500µlofcold70%ethanol.
5. ResuspendtheRNAin20µlofDEPC-treatedwater.
6. StoretheRNAat-20°Cor-70°C.
This protocol is for the Purication of RNA Transcripts
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