RNA ISOLATION - (Jul/24/2001 )
I am trying to isolate total RNA from a gram negative mixotrophic bacterium using Guanidinium thiocyanate as a denaturant. I have prepared all the reagents by myself and I am not using any kit. I got good yeild of RNA (about 4microgram per microlitre), the ratio of the absorbance at 260/280 was 1.937. When I ran about 20 micrograms on the denaturing gel (using MOPS buffer), I couldn't see 23S or 16S rRNA bands, only tRNA band was there (size about 200 nucleotides). There was no smear. Would you give me some suggestion? Thanks.
OD ratio does not mean anything but purity. RNA integration isthe key. You need to clean everything and make everything freeof RNase.