Dcr-2and other mutations were generated using a standard PCR-based cloning strategy and cloned into the corresponding vectors, and their identities were confirmed by sequencing analysis.or its mutants was expressed using the Bac-to-Bac baculovirus expression system in sf9 cells at 27 °C. One litre of cells was infected with 20 ml baculovirus at 27 °C. After growth at 27 °C for 48 h, the cells were collected, resuspended in buffer A with 0.
at a final concentration of 20 mM was added to the samples containing palindromic transcripts. The samples were heated to 95 °C for 5 min and then slowly cooled to room temperature. The annealed transcripts were purified by 8% denaturing urea PAGE, eluted from gel slices and precipitated with isopropanol. After centrifugation, the RNA precipitant was collected, washed twice with 70% ethanol and air-dried, and the RNA was dissolved in ultrapure water.
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