RNA extraction from E. coli
An.Huang
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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.
Steps
Preparation for experiment
Grow an overnight bacterial culture in the appropriate media at an appropriate temperature.
In the following day, take 1mLfrom overnight culture and add into 10mL LB media. Grow until the OD600 reads at 0.6-1.0.
RNA extraction
Harvest 1.5 mL culture (< 5x10%) by centrifugation at 3.000rpm for 10 min in a 1.5 mL microcentrifuge tube.
Discard all supernatant.
Resuspend the pellet in100µLfreshly 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 13.000rpm. Discard the DNA Clearance column and save the flow-through.
Transfer flow-through to the RNA binding column. Add 0.5 volume 100% ethanol to the lysate.
Centrifuge at 13000rpm. Discard the collection tube with the flow through and put the column back to a new collection tube.
Add 500µL Buffer RB to the column and centrifuge at 13.000rpm. Discard the flow-through.
Add another 500µL RNA Wash Buffer to the column and centrifuge at 13000rpm. Discard the flow-through.
Add another 500µL RNA Wash Buffer to the column and centrifuge at 13000rpm. Discard the flow-through and collection tube, put the column into a new collection tube.
Centrifuge the column at 13000rpm,0h 0m 0s, with the lid open, for another 0h 1m 0s.
Place the column to a RNase-free 1.5 mL tube, add 50-100 μL DEPC treated ddH2O to the column and centrifuge at 13000rpm.
Store the RNA solution at -20°C.
