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