Hello all,
I have been trying to grow E. coli cultures for the past few weeks but every time I extract total RNA, it does not show the 23S and 16S rRNA bands on the agarose gel. I have tried multiple times and reduced the incubation time from overnight to 6 hours. I can't tell if my E. coli is in lag phase or if I have contamination causing RNA degradation. Any help is appreciated.
Yes, you obviously have RNA degradation. Almost all your RNA is tiny, below the ladder. This should not be so. You have a massive source of RNAse somewhere. Look at your process. It's big. It should not be hard to find.
What are you incubating your RNA in for 6 hours? Do you mean ethanol precipitation?
How do you extract your RNAs? Hot phenol (with subsequent phenol-chloroform until you remove all proteins) or trizol kits work well If you haven't tried yet.
This is clearly contamination, as suggested by other users. I would post your RNA extraction procedure for someone else or me to look over. Also I would discard all solutions/tips/tubes used for RNA preps and grab all new reagents.
Thank you all so much for your answers. It was not contamination. I reduced the incubation time and it works fine now! I was starting off from the stock solution and my culture went into the death or LTSP phase during overnight incubation. I made the mistake of not monitoring the OD.
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