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mRNA Purification (E2065)

Synthesized mRNA can be purified by LiCl precipitation, phenol:chloroform extraction followed by ethanol precipitation, or by using a spin column based method (e.g. Monarch RNA Cleanup Kits NEB #T2030, #T2040 or #T2050).

LiCl Precipitation

The kit includes LiCl solution for quick recovery of the synthesized mRNA. 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 mRNA is suitable for transfection and microinjection experiments.

  1. To the 20 µl transcription reaction, add 30 μl water and 25 μl LiCl solution, mix well.
  2. Incubate at –20°C for 30 minutes.
  3. Centrifuge at 4°C for 15 minutes at top speed to pellet the RNA.
  4. Remove the supernatant carefully.
  5. Rinse the pellet by adding 500 μl of cold 70% ethanol and centrifuge at 4°C for 10 minutes.
  6. Remove the ethanol carefully. Spin the tube briefly to bring down any liquid on the wall.
  7. Remove residual liquid carefully using a sharp tip (e.g., loading tip).
  8. Air dry the pellet and resuspend the mRNA in 50 μl of 0.1 mM EDTA or a suitable RNA storage solution.
  9. Heat the RNA at 65°C for 5-10 minutes to completely dissolve the RNA. Mix well.
  10. Store the RNA at –20°C or below.

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 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.
  2. Extract with an equal volume of 1:1 phenol:chloroform mixture, followed by two extractions with chloroform. Collect the aqueous phase and transfer it 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 carefully.
  5. Rinse the pellet by adding 500 μl of cold 70% ethanol and centrifuge at 4°C for 10 minutes.
  6. Remove the ethanol carefully. Spin the tube briefly to bring down any liquid on the wall.
  7. Remove residual liquid carefully using a sharp tip (e.g., loading tip).
  8. Air dry the pellet and resuspend the mRNA in 50 μl of 0.1 mM EDTA or a suitable RNA storage solution.
  9. Store the mRNA at –20°C or below.

Spin Column Purification

Spin columns will remove unincorporated nucleotides, proteins and salts. Please follow the manufacturer’s instructions. Extra column washes before elution may help minimizing binding reagent carry over.

Gel Purification

The mRNA synthesized using DNA template encoding a poly(A) tail A125 has a defined length. Therefore gel purification can be done if necessary.