I extracted total RNA from bacteria after exposure to lysozyme for 30 minutes (these bacteria are naturally resistant to lysozyme). The 23S/16S rRNA ratio > 2. However, these samples look degraded when I ran polyacrylamide gel electrophoresis (see picture below). What is the reason? Is it a degradation? Is there another explanation?
Thank you for your answer.
General rule of thumb: if you can see a band, your RNA isn't degraded.
RNAses are usually highly processive. If you have RNAse contamination (ANY RNAse contamination), then you won't see ribosomal bands: you'll see a smear (and usually, a smear at the bottom of your gel).
How are you loading your RNA? The gel looks both overloaded and insufficiently denaturing, which won't help with resolution. Also, how are you assessing 23/16 ratio?
John Hildyard Thank you. I loaded 10 µg total RNA per lane. Maybe it is too much. Before loading, I usually wash the wells with buffer.
The 23S/16S ratio was assessed by analyzing agarose gel image using MultiGauge program (it is approximate measuring).
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