Qiagen 1-3
RNeasy Midi/Maxi RNA Extraction
1. Harvest bacteria by centrifuging at 4˚C, 4000 x g for 15 minutes in a swinging basket
a. Do not use more than 1 x 1010 bacteria for the midi kit.
b. Do not use more than 5 x 1010 bacteria for the maxi kit.
2. Decant supernatant
3. Repeat spin for 5 minutes at 4˚C, 4000 x g
4. Aspirate to remove all remaining media.
5. Heat the centrifuge to 20-25˚C.
6. Loosen the bacterial pellet by flicking the bottom of the tube.
7. Resuspend the bacteria thoroughly in 1 mL of lysozyme-containing TE buffer.
8. Incubate at room temperature for:
a. 2-5 minutes for Gram-negative bacteria
b. 5-10 minutes for Gram-positive bacteria.
9. Add 2 mL of Buffer RLT.
a. Be sure that β-ME is added to Buffer RLT before use.
10. Mix thoroughly by vortexing or shaking vigorously.
11. Centrifuge the bacterial lysate for 5 minutes at 3000-5000 x g.
12. Respin if you notice the pellets are loose.
13. Carefully transfer the supernatant to a new tube by pipetting:
a. Use a 10-15 ml tube for the midi kit.
b. Use a 50 ml tube for the maxi kit.
c. Use only this supernatant (lysate) in subsequent steps.
14. One tube at a time.
15. Add ethanol (96-100%) to the lysate
a. See Table 12 for amount to use.
16. Mix thoroughly by shaking.
a. Do not centrifuge.
17. Apply the sample, including any precipitate that may have formed, to a provided column
a. Use a RNeasy midi column in a 15 ml centrifuge tube for the midi kit.
b. Use a RNeasy maxi column in a 50 ml centrifuge tube for the maxi kit.
c. Maximum loading volume is 4.0 ml for midi or 15 ml maxi.
d. If the volume exceeds maximum, load aliquots successively onto the RNeasy
column
18. Close the tube gently
19. Centrifuge for 5 minutes at 3000-5000 x g.
a. If the maximum amount of starting material is used, it may be necessary to
increase centrifugation time to 10 minutes in order to allow the lysate to
completely pass through the column.
20. Discard the flow-through.
21. Repeat steps 17-20 if multiple aliquots are used
22. Pipet Buffer RW1 into the RNeasy column.
a. Use 2.0 ml for the midi kit.
b. Use 7.5 ml for the maxi kit.
23. Centrifuge fir 5 min at 3000-5000 x g to wash
24. Discard the flow-through.
Qiagen 2-3
25. Mix DNase I into Buffer RDD:
a. Add 20 μl DNase I stock solution to 140 μl Buffer SDD for the midi kit.
b. Add 30 μl DNase I stock solution to 210 μl Buffer SDD for the maxi kit.
c. Buffer SDD is supplied with the RNase-Free DNase Set.
26. Mix by gently flicking the tube
27. Centrifuge briefly to collect residual liquid from the sides of the tube.
28. Pipet the total volume of the DNase I incubation mix directly onto the RNeasy silica-gel
membrane
a. Make sure to pipet the DNase I incubation mix directly onto the RNeasy silica-gel
membrane.
b. DNase digestion will be incomplete if part of the mix sticks to the walls of the Oring of the RNeasy column.
29. Place on the benchtop (20-30˚C) for 15 minutes.
30. Pipet Buffer RW1 into the RNeasy column
a. Use 2.0 ml for the midi kit.
b. Use 7.5 ml for the maxi kit.
31. Place on the benchtop for 5 minutes.
32. Centrifuge for 5 minutes at 3000-5000 x g.
33. Discard the flow-through.
8. Add Buffer RPE to the RNeasy column.
a. Use 2.5 ml for the midi kit.
b. Use 10 ml for the maxi kit.
c. Be sure that ethanol is added to Buffer RPE before use
9. Close the centrifuge tube gently
10. Centrifuge for 5 minutes at 3000-5000 x g to wash the column.
11. Discard the flow-through.
12. Add another Buffer RPE to the RNeasy column
a. Use 2.5 ml for the midi kit.
b. Use 10 ml for the maxi kit.
13. Close the centrifuge tube gently
14. Centrifuge for 10 min at 3000-5000 x g to dry the RNeasy silica-gel membrane.
15. Dump out flow-through.
16. Spin empty tube 10 minutes more.
17. Remove the RNeasy column from the centrifuge tube carefully so the column does not
contact the flow-through.
18. Transfer the RNeasy column to a new supplied collection tube.
a. Use a 15 ml tube for the midi kit.
b. Use a 50 ml tube for the maxi kit.
19. Pipet the appropriate volume of RNase-free water directly onto the RNeasy silica-gel
membrane.
a. See Table 13 for amount to use.
20. Close the tube gently.
21. Let it stand for 1 minute
22. Centrifuge for 3 minutes at 3000-5000 x g.
23. Repeat elution steps 19-22.
24. Divide into about 50 ul aliquots for storage at -70˚C.
Qiagen 3-3
Table 12. Buffer volumes for RNeasy Midi/Maxi isolation of total RNA from bacteria
RNeasy
Number of
TE Buffer +
Buffer RLT
Ethanol (96Column
bacteria
lysozyme (ml)
(ml)
100%) (ml)
Midi ▲
1.4
5 x 108 - 5 x 109
0.5
2.0
Midi ▲
2.8
5 x 109 - 1 x 1010
1.0
4.0
Maxi ●
5.5
5 x 109 – 2.5 x 1010
2.0
7.5
4.0
15.0
Maxi ●
11.0
2.5 x 1010 - 5 x 1010
Table 13. RNase-free water volumes for RNeasy Midi/Maxi elution
RNeasy Column
Expected total RNA yield
RNase-free water
Midi ▲
Midi ▲
< 150 μg
150 μg – 1 mg
150 μl
250 μl
Maxi ●
< 1 mg
0.6 ml
Maxi ●
1-6 mg
1.2 ml