Please find attached a photo of the problem; After a simple Agarose gel electrophoresis of DNA with EtBr i find the gel "divided" in two parts. Analizing at the gel at 259nm it seems that the first part nearer to the dwells is more whitish while the second part seems "black" as aspected.
I Hope that the question is sufficiently clear, ut looking at the ohoto you will have a more clear vision of the problem.
Any suggestion on which could be the problem( maybe light-scattering.....)?
Any suggestion on how to solve it?
thanks for any advice and the help will be very appreciated
EtBr does move through the gel if you run the gel for a long time and if there is no etbr in the running buffer leading to a dark region at the bottom of the gel. You can still see your dna but if you think that you are missing any dna bands you can take the gel out and sit it in buffer with etbr for 20mins then the whole gel will have a brighter background but you are seeing your dna so what you are doing is working
Dear Daniele Brignole
I would like to draw your attention to some factors:
- It might have arised due to a malfunction of the camera of the gel documentation software. Try the gel imager using a white paper and fix if there is any problem regarding projection of the gel imager. Also, I would recommend you to check the gel under UV transilluminator before using the documentation software.
- The problem might have occurred due to the non-homogeneous dispersion of EtBr in the agarose gel. You should add EtBr when it comes to hand bearable temperature (approximately 45° C) and mix it vigorously before pouring into the gel caster.
All the best wishes for your success.
EtBr does move through the gel if you run the gel for a long time and if there is no etbr in the running buffer leading to a dark region at the bottom of the gel. You can still see your dna but if you think that you are missing any dna bands you can take the gel out and sit it in buffer with etbr for 20mins then the whole gel will have a brighter background but you are seeing your dna so what you are doing is working
thanks to all for the all the precious answers. i will try all the suggestions to see which fits better to our samples and gel.
Hello!
It is neither light scattering problem nor dye.
The amount of DNA present in those particular samples is very less compared to others that's why not looking brighter. EtBr luminescence is proportional to the quantity of DNA present in the sample loaded.
First of all your DNA is degraded highly. It is looking like a smear as if it is digested with some restriction enzyme. By the way, may I know which DNA it is!! If you mention what exactly you want to do with your DNA, I can advise.
Even the DNA is degraded it may work for PCR.
Good Luck!
Jetty Ramadevi I agree with you. Are you doing sonication of DNA? Otherwise you might want to fix the DNA smearing problem on every lane first as this is a more serious problem than the dark region you have. Every lane has a serious smear.
It is either caused by
1. Degradation of DNA
2. The gel is too hot
3. Too much DNA or size does not match the gel percentage
4. Slightly wrong buffer ion compared to gel
Yes, it may be true that it may be EtBr running out. However it should be happening throughout the gel instead of a single part of dark region since EtBr is neutrally charged. If you re-stain it and still no improvement, i suggest you to try other things.
This include:
1. Solving all of the above reasons
2. Try to use a different gel doc
3. Smaller gel to minimise reflection, plate the small gel in dark zone
4. Simply Invert the gel in the gel doc to see if it is refraction problem. If same pattern, with or without inverting then you know whether the pattern is on gel doc or the gel.
p.s. it is completely normal to have these patterns due to the placement of UV LED light probes in gel doc. I also have some dark and bright region for larger gels. Don't worry.
Hi! Kenneth,
It is not my work. I answered the question asked by Daniele.
He asked for suggestions what could be the reason, I have given my view.
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