Qiagen 1-2
RNeasy Mini RNA Extraction
Only do 2 or 3 tubes at a time
1. Harvest bacteria by centrifuging at 4000 x g for 5 minutes at 4˚C.
a. Do not use more than 1 x 109 bacteria.
2. Decant supernatant
3. Spin 5-10 minutes to remove all remaining media.
4. Loosen the bacterial pellet by flicking the bottom of the tube.
5. Resuspend the bacteria thoroughly in 100 μl of lysozyme-containing TE buffer by
vortexing.
a. See table below for amount of lysozyme.
6. Incubate at room temperature
a. See table below for incubation time.
Lysozyme concentration
Incubation time
in TE buffer
(room temperature)
Gram-negative bacteria
400 μg/ml
3-5 minutes
Gram-positive bacteria
3 mg/ml
5-10 minutes
7. Add 350 μl Buffer RLT to the sample.
a. Be sure that β-ME is added to Buffer RLT before use.
8. Mix thoroughly by vortexing vigorously.
9. If insoluble material is visible, centrifuge for 2 min in a microcentrifuge at maximum
speed
a. Use only the supernatant in subsequent steps.
10. Add 250 μl ethanol (96-100%) to the lysate.
11. Mix thoroughly by pipetting.
a. A precipitate may form after the addition of ethanol, but this will not affect the
RNeasy procedure.
b. Do not centrifuge.
12. Apply the sample (usually 700 μl), including any precipitate that may have formed, to an
RNeasy mini column placed in a 2 ml collection tube
13. Close the tube gently
14. Centrifuge for 15 seconds at about 8000 x g (about 10,000 rpm.)
15. Discard the flow-through.
16. If the volume exceeds 700 μl, load aliquots successively onto the RNeasy column and
repeat steps 12-15
17. Transfer the RNeasy column into a new 2 ml collection tube.
18. Pipet 500 μl Buffer RPE onto the RNeasy column.
a. Be sure that ethanol is added to Buffer RPE before use.
19. Close the tube gently
20. Centrifuge for 15 seconds at about 8000 x g (about 10,000 rpm) to wash the column.
21. Discard the flow-through.
22. Add another 500 μl Buffer RPE to the RNeasy column.
23. Close the tube gently
24. Centrifuge for 2 minutes at about 8000 x g (about 10,000 rpm) to dry the RNeasy silicagel membrane.
25. Place the RNeasy column in a new 2 ml collection tube
Qiagen 2-2
26. Discard the old collection tube with the flow-through.
27. Centrifuge in a microcentrifuge at full speed for 5 minutes.
28. To elute, transfer the RNeasy column to a new 1.5 ml collection tube.
29. Pipet 30-50 μl RNase-free water directly onto the RNeasy silica-gel membrane.
30. Close the tube gently
31. Centrifuge for 1 minute at about 8000 x g (about 10,000 rpm) to elute.
32. Repeat steps 29-31 using the first elution.
33. DNase treat at this step if needed
34. Store at -70˚C