Selected article for: "1x reaction buffer and RNA dependent RNA polymerase"
Author: Steffen Jockusch; Chuanjuan Tao; Xiaoxu Li; Thomas K. Anderson; Minchen Chien; Shiv Kumar; James J. Russo; Robert Kirchdoerfer; Jingyue Ju
Title: Triphosphates of the Two Components in DESCOVY and TRUVADA are Inhibitors of the SARS-CoV-2 Polymerase
  • Document date: 2020_4_5
    • ID: awbcw3gq_9
    Snippet: Extension reactions with RNA-dependent RNA polymerase. Oligonucleotides were purchased from Integrated DNA Technologies. The primer and template (sequences shown in Fig. 2) were annealed by heating to 70°C for 10 min and cooling to room temperature in 1x reaction buffer. The RNA polymerase mixture consisting of 6 µM nsp12 and 18 µM each of cofactors nsp7 and nsp8 (1:3:3 ratio) was incubated for 15 min at room temperature in 1x reaction buffer......
    Document: Extension reactions with RNA-dependent RNA polymerase. Oligonucleotides were purchased from Integrated DNA Technologies. The primer and template (sequences shown in Fig. 2) were annealed by heating to 70°C for 10 min and cooling to room temperature in 1x reaction buffer. The RNA polymerase mixture consisting of 6 µM nsp12 and 18 µM each of cofactors nsp7 and nsp8 (1:3:3 ratio) was incubated for 15 min at room temperature in 1x reaction buffer. Then 5 µl of the annealed template primer solution containing 2 µM template and 1.7 µM primer in 1x reaction buffer was added to 10 µl of the RNA polymerase mixture and incubated for an additional 10 min at room temperature. Finally, 5 µl of a solution containing either 2 mM TFV-DP and 20 µM UTP (a) or 2 mM Ec-TP (Toronto Research Chemicals), 200 µM UTP and 200 µM ATP (b) in 1x reaction buffer was added, and incubation was carried out for 2 hrs at 30°C. The final concentrations of reagents in the 20 µl extension reactions were 3 µM nsp12, 9 µM nsp7, 9 µM nsp8, 425 nM RNA primer, 500 nM RNA template, either 500 µM TFV-DP / 5 µM UTP or 500 µM

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