When purifying my DNA amplicons, I need to excise my DNA of interest from my gel (1.2% agarose).
I've observed that the DNA is distributed top and bottom of the thickness. Out of curiosity, I've done some research into why, but I can't find it. Does anyone have the answer?
Sadly I've none. I'll take illustrations next time I've to trimme a gel (in a few days).
If I can further describe : I have a DNA band, for example at 500bp. I cut it from my gel and when I flip it on its side I can see DNA on the top and botton.
I don't know if it's more clear... I'll post pictures asap!
Ok Simon Van Laethem That is much clearer to me. When you load dna onto a gel you add loading buffer with sucrose or glycerol to make the sample dense so that it sits in the bottom of the well and does not migrate out of the well into the buffer. Thus most of the dna and loading buffer is at the bottom of the well and the dna stays low down in the gel hence you are seeing a dna band low down in the gel but no dna in the upper half of the gel.
You also say that you see dna on the top of the gel. I think that this may be an artifact and not dna. Possibly we are seeing a reflection of UV light off the slightly more dense skin on the top of the gel or maybe if you are re using old buffer in the electrophoresis tank there is some diffusion of rna or dna from other users ( the bottom of the gel is protected by the gel tray ) and youare seeing diffused dna,rna or protein. If this is the case it should be tha same over the entire top of the gel so cutting a lice from an unused part of the gel should also show this top effect but not the bottom effect
Hello again,
Enclosed 4 pictures:
- Pic1: DNA band flipped on its side
- Pic2: "Clear fragment" flipped on its side
- Pic3: Step of my ladder flipped on its side
- Pic4: Localisation of the different fragment. Red = Pic1, green = Pic2, bleu = Pic3
I'm not sure about the theory of reflection, since the top band is not present on the "Clear fragment" nor in the ladder. This second band is really only present for DNA bands of interest (also at the smear level).
My buffer isn't the problem as it occurs also with brand new one.
I've been thinking about it since I started this topic, but I still don't have a new hypothesis.
PS: Sorry for the low quality of the pictures I made that quickly.
Thank you for the pictures . Simon Van Laethem . I agree with your analysis that the clear and ladder areas do not show either trapped fluorescence or reflected white light so it does seem that the buffer is not the cause and neither is it reflection as these would be found all over the gel, I cannot see any good explanation for this effect. Sorry, Paul
I think this is due to contact zones of the gel and a buffer which may create conditions slightly different than internal layer, such as ions..
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