Thermo Fisher Scientific Purification of RNA Transcripts User guide

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