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  • Purification of Synthesized RNA (E2040)

    Introduction

    Phenol-chloroform Extraction and Ethanol Precipitation

    For removal of proteins and most of the free nucleotides, phenol: chloroform extraction and ethanol precipitation of RNA transcripts is the preferred method.

    1. Adjust the reaction volume to 180 μl by adding 160 μl nuclease-free water. Add 20 μl of 3 M sodium acetate, pH 5.2 or 20 μl of 5 M ammonium acetate, mix thoroughly.
    2. Extract with an equal volume of 1:1 phenol/chloroform mixture, followed by two extractions with 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 minutes 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 50 μl of 0.1 mM EDTA. Store the RNA at -20°C or below.

    Protocol

    1. Phenol-chloroform Extraction and Ethanol Precipitation

      For removal of proteins and most of the free nucleotides, phenol: chloroform extraction and ethanol precipitation of RNA transcripts is the preferred method.
      1. Adjust the reaction volume to 180 μl by adding 160 μl nuclease-free water. Add 20 μl of 3 M sodium acetate, pH 5.2 or 20 μl of 5 M ammonium acetate, mix thoroughly.
      2. Extract with an equal volume of 1:1 phenol/chloroform mixture, followed by two extractions with 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 minutes 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 50 μl of 0.1 mM EDTA. Store the RNA at -20°C or below.
    2. Spin Column Chromatography

      Spin columns will remove unincorporated nucleotides, proteins and salts. Adjust the volume of the reaction mixture to 100 μl by adding 80 μl nuclease-free water, mix well. Purify the RNA by following the manufacturer’s instructions. Each reaction could produce up to 180 μg of RNA which may exceed column capacity thus requiring additional columns.

    3. Gel Purification

      When high purity RNA transcript is desired (e.g. labeled RNA probes for RNase protection assay or foot printing experiments), we recommend gel purification of the transcription product.