totalRna Extraction from Salmonella
nospam at nospam.edu
Wed Nov 11 11:41:43 EST 1998
In article <3649F9BA.E2A00332 at email.unc.edu>, Audrey Caro
<acaro at email.unc.edu> wrote:
> I had trouble to isolate large quatity of total RNA from Salmonella,
> compared with E. coli. I have tried two kits, the one from Bio 101 (for
> bacteria) and the one from Biotex (Rna Tack). I 've tried to extract
> total RNA from 20ml of an overnight culture in TS broth. How can I be
> sure that the lysis is correct? I tried once to add some lysosyme and
> the result was not better. Thanks for your suggestion.
> Audrey CARO
Yep, isolating RNA from Salmonella is a pain in the hiney. The best luck
I have had is using a SDS/boiling + Phenol extractions (lots of phenol,
did I mention phenol). I get great honking flocculent streamers in the
organic phase when doing extractions. Maybe some sort of polysaccharide.
I usually get tired after the sixth extraction. Although I generally get
decent yields of good RNA. ie. 1.2-1.5 mg RNA/10 ml mid log culture.
The 260/280 ratio was ~1.6, a little low, but the aliquoted RNA was stable
for at least a year at -70 C. If you want, I can send you the protocol.
Also, Salmonella 23S RNA is in two bits, so you have three rRNA bands on
your gel. Freaked me out the first time I ran a gel.
I have tinkered around with Qiagen RNAeasy, I used lysozyme, got RNA out,
although the yield was nothing to write home about. I do add the caveat
that it was a look-see experiment and I haven't tried to optimize the
Brad Nicholson |"If it worked the first time,
Department of Pathology | it wouldn't be research."
University of Utah |
Salt Lake City, UT 84132 | My opinions are solely my own.
Brad_Nicholson at hlthsci.med.utah.edu |
or: (801)-581-4901 | iligitimi non corborundrum
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