I performed RNA isolation from infected wheat tissue using NucleoZOL, followed by agarose gel electrophoresis. Although I stained the gel with ethidium bromide (EtBr), I observed blue fluorescence in certain regions (see attached image).
Experimental details:
- Sample: 20-30 mg infected wheat tissue
- Isolation: NucleoZOL protocol
- Gel: 1% agarose, stained with EtBr
- Buffer: TAE
- Imaging: UV transilluminator
What could be the cause of this blue fluorescence? Could it be due to secondary metabolites or residual compounds in the sample? Has anyone encountered a similar issue before?
I would appreciate any insights or suggestions. Thank you in advance!
Hello,
You mentioned that you pre-stained your gel with EtBr. EtBr is neutral in charge; however, in solution EtBr is positively charged and migrates to negative electrode in contrast to DNA, which is positively charged. That is why a blue hue of fluorescence signal is commonly observed on the top of the gel. Also remember that some coloured objects like paper, pinkish plastic covers also shine under UV light.
Best regards.
The blue diffuse band across the gel looks like one of the UV strip lights is failing and emitting visible light as well as UV light. You can short term move the gel to another position on the transilluminator where the gel does not sit over this bulb. Also short term you could get an electrician to move the dying bulb to the top of the array making more of the filter available but you really need to buy a new UV light and replace the dying bulb
one of your UV bulb is dead or you have exposed your gel too much and yopu see the image of thge transilluminator
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