Need Input on Northern With Bacterial RNA
mjward at vt.edu
Sun Mar 12 14:31:02 EST 1995
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.
College of Veterinary Medicine
Center for Molecular Medicine and Infectious Diseases
mjward at vt.edu
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