protocol for mRNA extraction from E. coli

Chi-Kuang Wen cwen at aux.btny.purdue.edu
Mon Apr 8 22:53:08 EST 1996


Jim wrote:
> 
> The procedure that I have used routinely with excellent results is in an
> article by Jinks-Robertson, et al. Cell 33, 865-876 (1983)
> 
> Basicly you lyse them _rapidly_ in boiling 1% SDS, then phenol extract 2x
> in hot phenol (be careful) and ppt with IsOH/NaAc. The RNA produced is
> very stable if you are clean enough (i.e. not messy). This will isolate
> total RNA and usually has a very low level of DNA contamination, if done
> exactly as the article states.
> 
> Just keep in mind that the average mRNA half life is about 2 1/2 minutes!
> Whatever technique you use had better be fast...
> 
> Jim
> 
> In article <4k05i9$hgi at hydra.acs.ttu.edu>, z3c20 at ttacs1.ttu.edu (xiaoming
> yi) wrote:
> 
> Hi, netters,
> 
> Can someone give me a GOOD reference on total mRNA extraction from E. coli?  I
> did not find such a protocol in "Molecular coloning" even there are several
> protocols for total mRNA extraction from mammalian cell cultures and alike.
> 
> Thank you,
> 
> Xiaoming Yi
> Texas Tech UniversityI guess that in "Methods in Enzomology" there are several articles 
describing separation of mRNA from animal and bacterial polysomes by 
sucrose gradient.  The idea is that you need to isolate the polysomes 
and separate the mRNA by sucrose gradient.  The rRNA is 28S/18S for 
eukaryotic cells and 23S/16S for prokaryotic cells.  The mRNA wouold be 
9S.  After another two enrichment steps, you may get mRNA from 
prokaryotic or eukaryotic cells.

regards,

Chi-Kuang


















































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