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bacterial RNA isolation

"David Alexander dalex at nexus.microimm.mcgill.ca
Thu Mar 30 16:33:46 EST 2000


Hi there,

I have recently done some bacterial (E.coli) RNA work
and used three methods to isolate the RNA.

Trizol does not work for E. coli.
I have not been able to determine the reason,
but I suspect it has something to do with poor cell lysis.

Although it is time consuming, the best method
(i.e. high yield and purity of RNA) uses CsCl gradients
I followed the protocol in Unit 4.2 of the Red Book/
Current Protocols in Molecular Biology.

Finally, I have used a shorter method (listed below),
which is similar to the bacterial RNA methods in Unit 4.4 of the Red 
Book. I was given this protocol by a group using microarrays and 
Mycobacterium, so it should work for you.
Just scale up the procedure for higher yields.

Good luck,

David.

----- 

RNA Extraction

Solutions
(I treat all solutions, tips, tubes, etc. with DEPC)

TSM = 10 uM Tris-HCl (pH 7.4),  140 uM NaCL, 1.5 mM MgCl2
5% NP-40 = nonidet P-40 (nonionic detergent) in water 
TSE+S = 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA
just before use, add SDS to 2%

Method:

1 - spin down cells (2 mL in a Eppendorf-type tube)
2 - discard supernatent, resuspend pellet in TSM + 0.5% NP-40
     (i.e. 270 uL TSM + 30 uL 5% NP 40)
3 - vortex, then place on ice for 2-3 minutes
4 - spin 10 seconds (doesn't sound like much, but it is enough)
5 - transfer supernatent to new tube
6 - add 1 volume (i.e. 300 uL) TSE+S to supernatent
7 - mix !
8 - add 2 volumes phenol:chloroform:isoamyl alcohol (25:24:1)
9 - vortex, then spin 5 minutes at 13 k
10 - transfer  supernatent to new tube 
(repeat steps 8, 9, 10)
11 - add 2 volumes chloroform:isoamyl alcohol (24:1)
12 - vortex, then spin 5 minutes at 13 k
13 - transfer superatent to a new tube and EtOH ppte
(i.e. 2.5 volumes 100% EtOH, 0.1 volume 3M NaOAc,
 precipitate at -20, -70, RT : whatever works for you
 spin 15 minutes, wash with 70% EtOH, and dry)
14 - resuspend pellet in 40 uL 1xTE (+ 0.1% SDS), store at -70 C
---




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