Analysis of oligonucleotides and unincorporated nucleotides from in vitro transcription by capillary electrophoresis in Pluronic F127 gels

Small functional RNAs required for structure studies are often prepared by in vitro transcription. Capillary electrophoresis in liquid crystalline gels of Pluronic F127 was used to analyze unfractionated in vitro transcription reactions and anion-exchange high-performance liquid chromatography (HPLC) fractions from transcription reactions. Guanosine monophosphate (GMP), the four nucleoside triphosphates (NTPs), abortive transcripts, and transcripts with lengths near the desired product length were simultaneously resolved and quantified in a single run. Oligonucleotides up to at least 35 nucleotides were resolved to baseline within 10 min using a moderate field (185 V/cm) and short effective capillary length (7.6 cm) for electrophoresis in 20% Pluronic F127 at pH 8.3 in Tris-borate-EDTA (TBE) buffer (30°C). Nucleotide migration times were 4—5 min, in the order UTP+CTP (unresolved)