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name="ProtocolList" |
Isolation of dsRNA from bacteria
Materials and Reagents |
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Equipment: |
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- A bench top centrifuge. Refrigerated centrifuge at 4°C is recommended.
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- Bench top refrigerated centrifuge at 4°C.
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Solutions:
- RNase free water
- 1x PBS (prepared with RNase free water)
- 150mM NaCl (prepared with RNase free water)
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- DNase 1 solution for on-column digestion (from Zymo, cat #E1010) (Optional)
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- Ethanol, 95-100% (from Biosci store is fine)
- Ethanol, 75% (diluted with 1x PBS)
- Ethanol, 70% in RNase free water.
Protocol |
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- 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.
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- 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.
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- Incubate sample at room temperature for 5min to fix cells.
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- Pellet the sample by centrifuging at 3500 x g for 5min at 4C.
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- Pellet the sample by centrifuging at 3500 x g for 5min.
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- 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.
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- 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.
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- 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.
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- 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.
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- 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.
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- 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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05-01-2024 |
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