Leang, Ching 1-1
RNeasy Mini Protocol for the Isolation of Total RNA from Bacteria
1. Harvest cells at 4 C.
2. Discard supernatant ensuring all media is completely removed.
3. Resuspend cells in 100 l lysozyme-containing TE buffer by vortexing.
a. For Gram Negative bacteria use 400 g/ml of lysozyme
b. Add 0.8 l of 50 mg/ml lysozyme in 100 l TE
4. Add 350 l Buffer RLT
a. Be sure -ME is added before use
b. 10 l -ME in 1ml RLT
5. Vortex vigorously.
6. If more than 5 x 108 cells are being processed, further homogenization with a
syringe and needle may increase yield.
7. Add 250 l ethanol (96-100%)
8. Mix well by pipeting.
a. Do not centrifuge.
9. Apply sample (usually 700 l), including any precipitate which may have
formed, to an RNeasy column.
10. Centrifuge for 15 seconds at 8,000 g ( 10,000 rpm).
11. Add 700 l Buffer RW1 onto the column
12. Centrifuge for 15 seconds at 8,000 g ( 10,000 rpm) to wash.
13. Discard flow-through and collection tubes.
14. Add 500 l Buffer RPE
15. Centrifuge for 15 seconds at 8,000 g ( 10,000 rpm).
16. Discard the flow-through
a. Reuse the collection tube.
17. Add 500 l Buffer RPE
18. Centrifuge for 2 minutes at maximum speed.
19. Place the column in a new 2-ml collection tube
20. Centrifuge at full speed for 1 minute.
21. Transfer column to 1.5ml tube
22. Add 30-50 l of RNase-free water directly onto the membrane.
23. Centrfuge for 1 minute at 8,000 g ( 10,000 rpm) to elute.
24. Repeat elution steps using first elution volume to re-elute, if the expected
RNA yield is > 30 g
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