---+ Isolation of dsRNA from bacteria ---+++ Materials and Reagents Use filter tips. Keep all solutions, reagents and plastics to be used for RNA work separate from general supplies. Solutions need to be certified RNase free. Equipment: * A bench top centrifuge. Refrigerated centrifuge at 4°C is recommended. * Nanodrop or Qubit Solutions: * RNase free water * 1x PBS (prepared with RNase free water) * 150mM NaCl (prepared with RNase free water) * DNase 1 solution for on-column digestion (from Zymo, cat #E1010) (Optional) * 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 from a saturated 1mL liquid culture (e.g. E. coli) or from resuspended cells grown on an agar plate (e.g. S. alvi). 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. If necessary, pipette up and down 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 at 4C. * 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 5000g for 5min. * Carefully collect the supernant and transfer it to a new tube. * Add 1mL of 95-100% ethanol 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 RNA concenttration in Nanodrop. Store sample at -80C for further aplications. * For visualization, 3-5uL of the sample can be run on a 1.2% agarose electrophoresis. 05-01-2024
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Topic revision: r4 - 2024-05-01 - LucioNavarro