Editing Cleancap mRNA Invitro Transcription (section)

=== RNA purification using anion-exchange chromatography ===

RNA from IVT can be purified using anion exchange chromatography on a 22 × 250 mm DNAPac PA100 column (Dionex) operated at 80°C.&nbsp;[https://www.thermofisher.com/order/catalog/product/043011#/043011 https://www.thermofisher.com/order/catalog/product/043011#/043011]

The column is run in buffers containing 20 mM Tris pH 8.0 and 5 M Urea (pH adjusted to 8.0 at room temperature). RNA of different lengths is eluted with a NaCl gradient. Salt gradients can be designed according to the respective RNA lengths to be separated. Short RNAs (2–10 nt) typically elute between 50 and 200 mM NaCl while longer constructs (60–80 nt) elute at 300–400 mM NaCl. RNA containing fractions can be analyzed using urea-PAGE, pooled and precipitated&nbsp; as below. After precipitation, RNAs can be desalted using a prepacked PD-10 Desalting column (GE Healthcare) and subsequently concentrated using a speedvac device.