Hi, I was trying to follow the RNA extension assay from this paper(Hillen, Hauke S et al. “Structure of replicating SARS-CoV-2 polymerase.” Nature vol. 584,7819 (2020): 154-156. doi:10.1038/s41586-020-2368-8), in Method section it says:

RNA was annealed in 50 mM NaCl and 10 mM Na-HEPES pH 7.5 by heating the solution to 75 °C and gradually cooling to 4 °C. RNA extension reactions contained RNA (5 μM), nsp12 (5 μM), nsp8 (15 μM) and nsp7 (15 μM) in 100 mM NaCl, 20 mM Na-HEPES pH 7.5, 5% (v/v) glycerol, 10 mM MgCl2 and 5 mM β-mercaptoethanol. Reactions were incubated at 37 °C for 5 min and the RNA extension was initiated by addition of NTPs (150 μM UTP, GTP and CTP, and 300 μM ATP). Reactions were stopped by the addition of 2× stop buffer (7 M urea, 50 mM EDTA pH 8.0, 1× TBE buffer).

My experimental condition was performed with some changes, but I don't think these these changes can lead to failure of this assay. My condition was:

RNA was annealed in 50 mM NaCl and 10 mM Tris pH 8 by heating the solution to 75 °C and gradually cooling to 4 °C. RNA extension reactions contained RNA (1 μM), nsp12, nsp8, nsp7 in 50 mM NaCl, 25 mM Tris pH 8, 1 mM NTPs, 5 mM MgCl2 and 1 mM DTT.

Reactions were incubated at 37 °C for 30 min. Reactions were stopped by the addition of 2× stop buffer.

I have no idea why I get no enlongated RNA product, what is the possible reason? the polymerase? the annealing process? the conditions?