HPLC purification was performed following previously published methods [26 (link), 27 (link)]. Large scale reactions (transcription or ligation) were quenched by adding EDTA to 50 mM final concentration and filtered using a 0.2 μm cellulose acetate syringe filter.
The Dionex Ultimate 3000 UHPLC system (Thermo) was equipped as follows: DNAPac PA200 22x250 Preparative column, LPG-3400RS Pump, TCC-3000RS Column thermostat, AFC-3000 Fraction collector, VWD-3100 Detector. Buffer A: 20 mM sodium acetate, 20 mM sodium perchlorate, pH 6.5. Buffer B: 20 mM sodium acetate, 600 mM sodium perchlorate, pH 6.5. Buffers were degassed and filtered before use. Column temperature: 75°C. Flow rate: 8 mL/min. Injection loop: 5 mL. Pump sequences: 0–10 min: 0% buffer B, 10–40 min: elution gradient, 40–50 min: 100% buffer B, 50–60 min: 0% buffer B. Elution gradients: 5’-RNA (30 nt): 22–30% buffer B, 3’-RNA (16 nt): 15–25% buffer B, Full-length RNA (46 nt): 15–45% buffer B.
Collected fractions were tested for RNA of interest by loading 0.1 μL on a 20% denaturing PAGE.
The Dionex Ultimate 3000 UHPLC system (Thermo) was equipped as follows: DNAPac PA200 22x250 Preparative column, LPG-3400RS Pump, TCC-3000RS Column thermostat, AFC-3000 Fraction collector, VWD-3100 Detector. Buffer A: 20 mM sodium acetate, 20 mM sodium perchlorate, pH 6.5. Buffer B: 20 mM sodium acetate, 600 mM sodium perchlorate, pH 6.5. Buffers were degassed and filtered before use. Column temperature: 75°C. Flow rate: 8 mL/min. Injection loop: 5 mL. Pump sequences: 0–10 min: 0% buffer B, 10–40 min: elution gradient, 40–50 min: 100% buffer B, 50–60 min: 0% buffer B. Elution gradients: 5’-RNA (30 nt): 22–30% buffer B, 3’-RNA (16 nt): 15–25% buffer B, Full-length RNA (46 nt): 15–45% buffer B.
Collected fractions were tested for RNA of interest by loading 0.1 μL on a 20% denaturing PAGE.
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