Hello, I have problems with RNA isolation from gram positive bacteria producing EPS. I obtain a cloud instead of the classic clear rRNA bands. I tried with liquid nitrogen or beads to break cells and then phenol:isoamilic:chloroform or a commercial kit to extract. I always obtain the same result (a cloud in the photo). Does anyone have a protocol for gram positive mucoid bacteria?
With the amount of information provided it is hard to interpret your gel.
1. Try isolating RNA from other bacteria E. coli or something and compare if the results are similar. If they are, then the problem is not with Extracellular polymeric substances (EPS) but with your gel or your isolation method.
2. Run a DNA/RNA marker alongside the isolated RNA. If the signal close to the wells is your RNA (not DNA contamination) your isolation might be good. It is gels fault that it looks bad. Wells are narrow and not flat so most of your sample runs in the right and left corner of the well, which distorts how your bands look. Maybe the gel was used too early without giving it the time to solidify.
3. Last option, I guess you are using normal Agarose gel and TAE/TBE buffer. So, your sample might not be properly heated and denatured in RNA loading buffer (containing formamide) before loading onto the gel.
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