Oct 07, 2022
RNA extraction from E. coli
- 1XJTLU
Protocol Citation: An.Huang 2022. RNA extraction from E. coli. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw44wl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 06, 2022
Last Modified: October 07, 2022
Protocol Integer ID: 70925
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Abstract
RNA extraction is a fundamental step in multiple experiments, for example, qPCR. This protocol helps conduct a simple RNA extraction procedure.
Materials
Buffer LY (added 1% volume of dithiothreitol), Buffer RB, RNA Wash Buffer, DEPC-Treated ddH2O, RNA Columns, DNA Clearance Column, Collection Tubes, 1.5 mL RNase-free microfuge tube, Lysozyme buffer (0.4 mg/mL)
Preparation for experiment
Preparation for experiment
Grow an overnight bacterial culture in the appropriate media at an appropriate temperature.
In the following day, take 1 mL from overnight culture and add into 10 mL LB media. Grow until the OD600 reads at 0.6-1.0.
RNA extraction
RNA extraction
17m 30s
17m 30s
Harvest 1.5 mL culture (< 5x10%) by centrifugation at 3.000 rpm, 00:10:00 for 10 min in a 1.5 mL microcentrifuge tube.
10m
Discard all supernatant.
Note
You may use a pipette to remove the remaining liquid at the bottom of the tube.
Resuspend the pellet in100 µL freshly prepared Elution Buffer (10mM Tris-HCL pH 8.5) containing lysozyme (0.4 mg/mL lysozyme for Gram negative bacteria). Mix by tapping gently.
Incubate the resuspended pellet at room temperature for 3-5 min for Gram-negative bacteria.
Add 400 μL Buffer LY. Mix gently.
Transfer the cleared lysate to a DNA Clearance column pre-inserted in a 2 mL Collection Tube. Centrifuge at 13.000 rpm, 00:02:00 . Discard the DNA Clearance column and save the flow-through.
2m
Transfer flow-through to the RNA binding column. Add 0.5 volume 100% ethanol to the lysate.
Note
For example: 250 μL 100% ethanol for 500 μL.
Centrifuge at 13000 rpm, 00:01:00 . Discard the collection tube with the flow through and put the column back to a new collection tube.
1m
Add 500 µL Buffer RB to the column and centrifuge at 13.000 rpm, 00:00:30 . Discard the flow-through.
30s
Add another 500 µL RNA Wash Buffer to the column and centrifuge at 13000 rpm, 00:00:30 . Discard the flow-through.
Note
Ethanol should be first added into RNA Wash Buffer before use.
30s
Add another 500 µL RNA Wash Buffer to the column and centrifuge at 13000 rpm, 00:00:30 . Discard the flow-through and collection tube, put the column into a new collection tube.
30s
Centrifuge the column at 13000 rpm , with the lid open, for another 00:01:00 .
1m
Place the column to a RNase-free 1.5 mL tube, add 50-100 μL DEPC treated ddH2O to the column and centrifuge at 13000 rpm, 00:02:00 .
Note
The RNA is in the flow-through.
2m
Store the RNA solution at -20 °C .