Need Input on Northern With Bacterial RNA

Shahram Mori smori at nmsu.edu
Mon Mar 13 02:39:14 EST 1995


Christine Ward (mjward at vt.edu) wrote:
: Greetings Netters:

: I could really use some input from those of you with experience doing 
: Northerns on bacterial RNA.  I have been using the Chomczynski (sp?)
: and Sacchi procedure (AGPC procedure, Analytical Biochem.) to isolate 
: total RNA from a Gram negative organism.  I have upscaling the original
: procedure by a factor of 10 to isolate RNA from about 1 x 10e9 organisms.
: When I do my Northerns, all I see is one really good smear.

: When I stain the total RNA on the membrane with methylene blue, my markers 
: look OK, and I can clearly see the 23S and 16S rRNA bands in my RNA prep.
: I believe my technique is quite good--using clean pipettors, DEPC'd solutions, 
: storing RNA in formamide etc.  OD260/OD280 is about 1.8, which supposedly
: indicates "pure RNA".  My gut instinct is that I have some residual 
: protein (and therefore RNAse carryover), OR that the particular RNA I am 
: trying to detect has a very rapid turnover rate (even though the time going
: from the culture in the H2O bath to the guanidinium solution is less than 4
: minutes).  When I tried adding an additional phenol:CHCL3 step, I lost my 
: RNA.

: Does anyone know of any compounds I could add to my growing culture 
: to slow the RNA turnover rate without killing my bacteria?  Would 
: adding chloramphenicol or spectinomycin be reasonable to try?  
: I would appreciate if anyone who has any pearls of wisdom to offer
: regarding RNA isolation from bacteria would post some suggestions.
: Thanks.

: Christine Ward
: Virginia Tech 
: College of Veterinary Medicine
: Center for Molecular Medicine and Infectious Diseases
: Blacksburg, VA
: mjward at vt.edu

Christine,
If your method involved SSC or SSCP, the high salt will definitely kill 
any RNases that might be carried over. Also run your RNA on a gel. In 
addition to those bands you should see two more. If there is a bright 
smear on the bottom of the gel, you have degradation.   
Cheers
--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





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