Home Protocols Purification of Synthesized RNA (E2070)

Purification of Synthesized RNA (E2070)

In general, synthesized RNA can be purified by LiCl precipitation, phenol:chloroform extraction followed by ethanol precipitation, or by using a spin column method. If absolute full length RNA is required, we recommend gel purification.

LiCl Precipitation
The kit includes LiCl solution for rapid recovery of the synthesized RNA. LiCl is effective in removing the majority of unincorporated NTPs and enzymes. However, RNAs shorter than 300 nucleotides or at a concentration lower than 0.1 mg/ml do not precipitate well. In such cases, other purification methods may be used. LiCl purified RNA is suitable for cap addition with NEB’s Vaccinia Capping System (NEB #M2080) and Poly(A) tailing with NEB’s Poly(A) Polymerase (NEB #M0276).
  1. Adjust the reaction volume to 50 µl by adding nuclease-free water.
  2. Add 25 µl LiCl solution and mix well.
  3. Incubate at –20°C for 30 minutes.
  4. Centrifuge at 4°C for 15 minutes at top speed to pellet RNA.
  5. Remove the supernatant and rinse the pellet with 500 µl of ice cold 70% ethanol.
  6. Resuspend the RNA in 50 µl of 0.1 mM EDTA. Store the RNA at –20°C or below.

Phenol:chloroform Extraction and Ethanol Precipitation
For removal of proteins and most free nucleotides, phenol:chloroform extraction followed by ethanol precipitation of RNA transcripts is the preferred method.
  1. Adjust the reaction volume to 180 µl by adding nuclease-free water. Add 20 µl of 3 M sodium acetate, pH 5.2 or 20 µl 5 M ammonium acetate and 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 ice cold 70% ethanol.
  5. Resuspend the RNA in 50 µl 0.1 mM EDTA. Store the RNA at –20°C or below.
Spin Column Purification
Spin columns will remove unincorporated nucleotides, proteins and salts. Adjust the volume of the reaction to 100 µl by adding nuclease-free water and mix well. Purify the RNA by following the spin column manufacturer’s instructions. Each reaction produces ≥ 80 µg of RNA, which may exceed the column capacity, thus requiring additional columns.

Gel Purification
When high purity RNA transcripts are desired (RNA probes for footprinting assays or RNase protection assays), we recommend gel purification of the transcription product.