---+ Isolation of dsRNA from bacteria ---+++ Materials and Reagents This protocol can be used to validate expression of dsRNA in <i>E. coli</i> (e.g. <i>E.coli</i> HT115(DE3)) or in other engineered dsRNA-expressing bacteria (e.g <i>S. alvi</i> or <i>S. symbiotica</i>). Equipment: * Bench top centrifuge. * Bench top refrigerated centrifuge at 4°C. * Nanodrop or Qubit Solutions: * RNase free water * 1x PBS (prepared with RNase free water) * 150mM NaCl (prepared with RNase free water) * Ethanol, 95-100% (from Biosci store is fine) * Ethanol, 75% (diluted with 1x PBS) * Ethanol, 70% in RNase free water. ---+++ Protocol * This protocol starts with a pellet of bacteria cells. Depending on your experiment you will need to spin cells (3500 x g for 5min) from a saturated 1mL liquid culture (e.g. <i>E. coli</i> or <i>S. symbiotica</i>) or from resuspended cells grown on an agar plate (e.g. <i>S. alvi</i>). If the pellet comes from -80C storage, make sure it is totally thawed on ice before starting. * Resuspend cell pellet in 1mL of 75% ethanol (diluted with 1x PBS). If necessary, pipette up and down carefully to resuspend completely the cells. * Incubate sample at room temperature for 5min to fix cells. * Pellet the sample by centrifuging at 3500 x g for 5min. * Resuspend cell pellet in 300uL of 150mM NaCl. If necessary, pipette up and down to resuspend completely the cells. * Incubate sample at room temperature for 1 hour. * At this point, dsRNA should in solution along with cells and debris. To remove cells/debris, spin sample at 5000 x g for 5min. * Carefully without disturbing the cell pellet, collect the supernant and transfer it to a new tube. * Add 1mL of 95-100% ethanol to the collected supernatant and mix well by short vortex. * Incubate sample at -20C for at least 4 hours or overnight. * Centrifuge sample at max speed (>16,000 x g) for 15min at 4C. * Discard supernatant by pipetting up all liquid avoiding to disturb the whitish pellet at the bottom of the tube. * Wash the pellet with 1mL of 70% ethanol. * Centrifuge sample at max speed (>16,000 x g) for 5-10min at 4C. * Discard supernatant by pipetting up all liquid avoiding to disturb the whitish pellet at the bottom of the tube. * Let the tube lid open at 37C for 20-30min to dry dsRNA pellet. * Finally add 50uL of RNase free water and incubate for 15min at room temperature. Mix well by vortex during ~15s. * Measure dsRNA concentration in Nanodrop. Store sample at -80C for further aplications. * For visualization, run 3-5uL of the sample on 1.2% agarose electrophoresis.
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Topic revision: r5 - 2024-05-02 - LucioNavarro