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
: 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.
: Christine Ward
: Virginia Tech
: College of Veterinary Medicine
: Center for Molecular Medicine and Infectious Diseases
: Blacksburg, VA
:mjward at vt.edu
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.
Shahram Mori _/\_
Program in Molecular Biology _\ /_
Dept. of chemistry and Biochemistry Box 3C \_ _/
NMSU Las Cruces NM ||