RNAylation of SARS-CoV-2 nsp9 by nsp12 was performed in a final buffer containing 50 mM Tris, pH 7.5, 1 mM DTT, 1 mM MnCl2, 1 μg yeast inorganic pyrophosphatase (ThermoFisher EF0221), 20 μM nsp9 and 40 μM triphosphorylated RNA corresponding to the 5′ end of the SARS-CoV-2 genome (5′-pppAUUAAAGGUU-3′). The inhibitor was added at final concentrations of 1, 10 and 100 μM. Reactions were started through the addition of nsp12, final concentration 2 μM, and stopped after 30 min incubation at 25°C in 4X SDS loading die solution. Nsp9 (theoretical MW 12.4 kDa) and pRNA-nsp9 (theoretical MW 15.8 kDa) were separated on 4–20% gradient SDS-PAGE gels (NuSep), and stained with QuickBlue Protein Stain (Lubio Science). Gels were scanned on the Amersham ImageQuant800 machine to obtain density. Percent RNAylation of nsp9 was calculated as the ratio of the nsp9-pRNA10 band, relative to total nsp9 (nsp9 plus nsp9-pRNA10 bands). Inhibition of RNAylation was calculated as a percentage, relative to no inhibitor controls (n = 2).
Ef0221
Manufactured by Thermo Fisher Scientific
The EF0221 is a laboratory centrifuge designed for a variety of applications. It features a maximum speed of 6,000 rpm and a maximum relative centrifugal force (RCF) of 4,400 x g. The unit is equipped with a brushless motor and digital speed and time control.
Automatically generated - may contain errors
Lab products found in correlation
Malachite green phosphate assay kit, by Merck Group (2 mentions)
Imagequant 800, by Cytiva (1 mentions)
Inorganic pyrophosphatase, by Thermo Fisher Scientific (1 mentions)
Exo resistant random primer, by Thermo Fisher Scientific (1 mentions)
Phi29 dna polymerase, by Thermo Fisher Scientific (1 mentions)
Templiphi 100 amplification kit, by GE Healthcare (1 mentions)
Synergy mx, by Agilent Technologies (1 mentions)
4 protocols using ef0221
1
SARS-CoV-2 nsp9 RNAylation Assay
2
Random Rolling Circle Amplification
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Random rolling circle amplification was performed as previously described (7 (link)) with some modifications. A 20 μl reaction was set up using 5 μl of size-selected template DNA, 1 mM dNTPs, 10 U Phi29 DNA polymerase (EP0092, Thermo Fisher Scientific), 50 μM Exo-resistant random primer (SO181, Thermo Fisher Scientific), 0.02 U inorganic pyrophosphatase (EF0221, Thermo Fisher Scientific) and 1× Phi29 DNA polymerase buffer (supplied with enzyme). The reaction was run at 30°C for 18 h and stopped by heating to 65°C for 2 min. Product DNA was purified by sodium acetate/ethanol precipitation. We also used the illustra TempliPhi 100 amplification kit (25640010, GE Life Sciences) and obtained similar amplification results.
3
Quantifying CRISPR-Cas13a Collateral cOA Production
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The in vitro cOA detection assays were conducted in TtCmr activity assay buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM DTT, 1 mM ATP, and 1 mM MgCl2) to which Cmr-complex (62.5 nM final concentration) as well as the RNA substrates (200 nM, listed in Supplementary Table S1 ) were added. The reaction was incubated at 65 °C for 1 h after which 0.05 units of pyrophosphatase (ThermoFisher EF0221) were added, followed by an incubation at 25 °C for 30 min (Fig. 2 ). Alternatively, thermostable pyrophosphatase (NEB #M0296) was added during the 1 h incubation at 65 °C (Fig. 3 ). cOA quantification was achieved by using the Malachite Green Phosphate Assay Kit (Sigma-Aldrich MAK307). This resulted in an OD650 signal, which was measured on a BioTek Synergy Mx platereader using the BioTek Gen5 software.
The unitless relative PPi levels (cOA-production) presented in Figs.2 and 3 were calculated by expressing the OD650 signal from the individual RNA targets (containing the indicated mismatches) as a ratio of the OD650 signal obtained from the fully complementary target RNA.
The unitless relative PPi levels (cOA-production) presented in Figs.
4
CRISPR-Cas13 Cleavage Activity Assay
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The in vitro cOA detection assays were conducted in TtCmr activity assay buffer (20mM Tris-HCl pH 8.0, 150mM NaCl, 10mM DTT, 1 mM ATP, and 0.5 mM 30 MgCl2) to which Cmr-complex (62.5 nM) as well as the RNA substrates (200 nM, listed in Table S1) were added. The reaction was incubated at 65˚C for 1 hour after which 0.05 units of pyrophosphatase (ThermoFisher EF0221) was added, followed by an incubation at 25˚C for 30 minutes. Alternatively, thermostable pyrophosphatase (NEB #M0296) was added during the 1h incubation at 65˚C. The signal was visualized using the Malachite Green Phosphate Assay Kit (Sigma-Aldrich MAK307).
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