Isolation of dsRNA from bacteria

Materials and Reagents

This protocol can be used to validate expression of dsRNA in E. coli (e.g. E.coli HT115(DE3)) or in other engineered dsRNA-expressing bacteria (e.g S. alvi or S. symbiotica).

Equipment:

  • Bench top centrifuge.
  • Bench top refrigerated centrifuge at 4C.
  • 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. E. coli or S. symbiotica) 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 (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 - 01:48:59 - Main.LucioNavarro
 
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