Synthesized RNA can be purified by LiCl precipitation, phenol:chloroform
extraction followed by ethanol precipitation, or by using a spin column based
method. If absolute full length RNA is required, we recommend gel purification.
The kit includes LiCl solution for quick recovery of the synthesized RNA. LiCl
precipitation of RNA is effective in removing the majority of unincorporated
NTPs and enzymes. However, RNAs shorter than 300 bases or at concentrations
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
- Adjust the reaction volume to 50 μl by adding nuclease-free water.
- Add 25 μl LiCl solution and mix well.
- Incubate at –20°C for 30 minutes.
- Centrifuge at 4°C for 15 minutes at top speed to pellet the RNA.
- Remove the supernatant and rinse the pellet with 500 μl of ice cold 70%
- Resuspend the RNA in 50 μl of 0.1 mM EDTA. Store the RNA at –20°C or
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.
- 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 of 5 M ammonium acetate and mix thoroughly.
- 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.
- Precipitate the RNA by adding 2 volumes of ethanol. Incubate at –20°C for at least 30 minutes and collect the pellet by centrifugation.
- Remove the supernatant and rinse the pellet with 500 μl of ice cold 70% ethanol.
- 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 mixture 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 up to 200 μg of RNA, which may exceed column capacity, thus requiring additional columns.
When high purity RNA transcript is desired, we recommend gel purification of the transcription product.