| Assay Method Information | |
| | Polθ Polymerase Domain WT K, Assay |
| Description: | Assay measurements were performed with 1× buffer comprising 25 mM Tris pH 7.5, 12.5 mM NaCl, 0.5 mM NaCl, 5% (v/v) glycerol, 0.01% v/v Triton x-100, 0.1 mg/ml BSA, 1 mM DTT. Test compounds were prepared by dilution in 100% DMSO to give a 12 μM intermediate stock of each (100× final top concentration). 100 nL of 23×1:1.5-fold serial dilutions and a DMSO only control were dispensed using the Tecan dispenser into Greiner 384 well black low volume plates (product code 784076). DMSO concentration was maintained at 1% of the final assay volume by back filling with DMSO.2× Working stock of substrate (200 nM of DNA substrate and 100 μM dNTPs) and enzyme (0.312 nM Polθ) were made up in assay buffer. 5 μL/well of both enzyme and substrate 2× solutions were dispensed using a Tempest dispenser (Formulatrix) into assay plates that had been pre-dispensed with compound to give a final assay concentration of 100 nM DNA substrate, 50 μM dNTPs and 0.156 nM Polθ. To stop the reaction, 5 μL of a solution containing 25 mM Tris-HCl pH 7.5 and 20 mM EDTA was added at 6 timepoints (t=0, 15, 30, 60, 90, 120, 150, 180 mins) using the Tempest's time delay function. The plates were covered during the time course to prevent evaporation. After completing the assay, 5 μL of detection reagent (25 mM Tris-HCl pH 7.5 and 2.5% (v/v) PicoGreen) was dispensed into the wells using a Tempest liquid handler (Formulatrix) and plates subsequently read on the CLARIOstar Plus (BMG Labtech) using the default optical settings for fluorescein and the auto gain/focus settings. |
| Affinity data for this assay | |
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