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.
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