Barrick Lab :: DsRNAprepbacteria

---+ 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, 100% (from Biosci store is fine)
   * 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 (4000 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 250uL of 1x PBS. If necessary, pipette up and down carefully to resuspend completely the cells.
   * Add 750uL of 100% ethanol and mix well by short vortex.
   * Incubate sample at room temperature for 5min to fix cells.
   * Pellet the sample by centrifuging at 4000 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 10,000 x g for 5min.
   * Carefully without disturbing the cell pellet, collect the supernant and transfer it to a new tube. At this point, dsRNA should be in solution and can be visualized by 1.2% agarose electrophoresis (~10uL/well).

Optional: Higher concentration of dsRNA can be obtained by ethanol precipitation as follows.

   * Add 3x volumes of 100% ethanol (~1.0mL) to the collected supernatant from above 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 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.
Barrick Lab > ProtocolList > DsRNAprepbacteria
Contributors to this topic
LucioNavarro
Topic revision: r6 - 2025-03-20 - 21:09:26 - Main.LucioNavarro