Selected article for: "room temperature and tris buffer"
Author: Minchen Chien; Thomas K. Anderson; Steffen Jockusch; Chuanjuan Tao; Shiv Kumar; Xiaoxu Li; James J. Russo; Robert Kirchdoerfer; Jingyue Ju
Title: Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase
  • Document date: 2020_3_20
    • ID: 8prn86g6_12
    Snippet: Extension reactions with RNA-dependent RNA polymerase. Oligonucleotides were purchased from IDT, Inc. 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 was incubated for 15 min at room temperature in a 1:3:3 ratio in 1x reaction buffer. Then 5 µl of t.....
    Document: Extension reactions with RNA-dependent RNA polymerase. Oligonucleotides were purchased from IDT, Inc. 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 was incubated for 15 min at room temperature in a 1:3:3 ratio 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 2'-F,Me-UTP (a), 2 mM 3'-F-dTTP (b), 2 mM TFV-DP and 20 µM UTP (c) or 3'-N 3 -dTTP (d) 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 2'-F,Me-UTP (Sierra Bioresearch), 500 µM 3'-F-dTTP (Amersham Life Sciences), 500 µM TFV-DP/50 µM UTP or 500 µM 3'-N 3 -dTTP (Amersham Life Sciences), and 1x reaction buffer (10 mM Tris-HCl pH 8, 10 mM KCl, 2 mM MgCl 2 and 1 mM β-mercaptoethanol). Following desalting using an Oligo Clean & Concentrator (Zymo Research), the samples were subjected to MALDI-TOF-MS (Bruker ultrafleXtreme) analysis.

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