Strange question regarding DNA in gel-slice... - (Nov/11/2005 )
Hi,
I have a rather strange and possibly very stupid question... Today I ran a quite normal 1.5 % agarose gel (in 1xTAE) with some plasmid DNA cut with an restriction enzyme (3 µg of DNA cut in 50 µl). My gel was a big one; about 11x14 cm and ~10 mm thick (I placed all of my sample in one well). I need to isolate the linear plasmid, so after EtBr staining and destaining I cut the band from the gel under UV light. Then the puzzle starts... This gel slice was tilted and I noticed that there was a band on the top and bottom of the gel slice while the mid part was empty. So, I removed the mid part and took the stained parts... Was that a terrible mistake? Does the EtBr penetrate only inty the outermost parts of such a thick gel (stained for about 5 minutes) while DNA in the more central part of the gel doesn't get any stain? Or does the DNA for some reason place itself far up and far down in a thick gel so the gel is more or less empty in the middle??
I will, of course, find out next time just by restaining the whole gel slice after cutting it from the gel or by adding some EtBr in my sample before loading it onto the gel, but that answer is days ahead of me...
Anyone with an answer out there?
Thank's!
Sonja
If you didn't add EtBr to the gel when you poured it, and you stained in liquid solution after running, then I think that you did have DNA in that middle part, probably the "best" for downstream applications because it didn't have EtBr (darn murphys law! 
) it makes sense that the stain would only penetrate the outside parts of the gel especially with only 5 minutes....
) it makes sense that the stain would only penetrate the outside parts of the gel especially with only 5 minutes....Thank's for your reply, although it wasn't the answer I was hoping for... I'll just have to start all over again (I need as much of my vector as possible so throwing away half of the gel slice wasn't very smart...).
Thank's again- this discussion forum is brilliant
Sonja
darn ... I encountered that problem as well sometimes, but I never really though about it ... still your explanation sound quite logical to me ... damn 
Hi...
Well, I ran a new gel today and I'm not really convinced that I did such a big mistake after all. My gel was not so thick this time, and the wells were made with a relatively large distance from the bottom of the gel tray (I had to make sure that there would be no leakage when removing the comb where I had used tape to make larger wells). So, after EtBr staining of the gel in a EtBr-bath and then unstaining, I cut the gel slice and again tilted it just to have a look. This time I didn't remove any gel from the central part of the gel slice, but again there was staining only at the top of the gel (a distinct "line") and interestingly there was a very distinct line at the area representing the bottom of the wells. However, that band was in the middle of the gel due to my rather large "safe distance" when placing the comb in the gel. In other words, this time it could not be the lack of EtBr in any part of the gel causing these separated bands since one of them actually was more or less in the middle of the gel! So, after all, maybe this is just the way it looks even if I really don't understand why...
Sonja
Hi, so I think you are still seeing the same thing... Yes the EtBr gets into the gel, what happens is the DNA nearest the outside of the gel is taking the EtBr up...
Lets take the "bottom of the well" band... The EtBr migrates into the gel far enough to reach this DNA (obviously 
) What happens there is that the EtBr is getting intercalated into these DNA strands, so the DNA there is "soaking up" the EtBr and doesn't let it pass by to intercalate into the DNA in the middle of the gel. So it isn't that EtBr cant get into the gel, just that the DNA nearest the outside of the gel is like (if you will give me some metaphoric leeway) a sponge, you have to saturate this DNA with EtBr before it can get intercalated into the DNA at the middle...
I think this is probably what is going on, I hope this makes sense to you... You may ask, as I am wondering now, why the DNA in the middle of the band isn't picking up EtBr from the front and back side (ie the top and bottom of the lane), but this I just don't know...
Maybe someone else has an idea...
i have been discusing this with my labmates, and we are still unsure. to us this seems still the wrong explanation. the reasonis, that the bands are distinct. if they would faint towards the middle ... okay ... but they are perfectly stained, but distinct. to me, that leaves out the diffusion argument. i can't explain in other ways, but that leaves the up-to-now mentioned reasons senseless ...
any other ideas?
That is a good point... That second theory would suggest that there would be a fading band... I still have a problem with the other flaw in this theory... I still can't think of why there wouldn't be stains on the sides of the band facing the the top and bottom of the gel too...
Was the bottom of the well farther away from the top of the gel than the bottom of the top band?? (is the sentance confusing enough??) I mean to say is it possible the first theory still applies but the EtBr migrated further into the gel b/c it is thinner? This still doesnt explain why the bands are distinct but maybe something else accounts for that ?? 
What about a little piece of gel stuck in the well... I somtimes see this, it gets pulled up when you take out the comb... Unlikely to happen twice in a row I guess but maybe that could be the cause???
Hey, I just thought of something... is it possible your gel is melting a little in the middle? Would that explain this phenomenon??
The problem is I have looked at my gel slices from the side too, and I see one thick band and clear from the bottom of the well to the bottom of the gel... But I put EtBr in the gel...
okay one more, how full is the well, maybe there is like a surface tension keeping some near the top and the rest moves to the bottom?? What is the intensity of each band? does one seem to have more DNA than the other??
I like this puzzle... 
I hope it gets figured out.. Maybe someone out there with more experience can help...
Hi
You may have noticed that when you load a gel, the DNA/marker mix flows into the well and sits along the bottom and narrow sides of the well most, leaving a clear patch in the middle, which appears to be a sort of mixing problem. If you run a gel like this the DNA will run in this pattern, so to fix this give the DNA a gentle mix in the well so that the solution is evenly distributed over the large face of the well. Gives much better resoltion when using thick combs, and makes gel extraction much easier!
bob
You may have noticed that when you load a gel, the DNA/marker mix flows into the well and sits along the bottom and narrow sides of the well most, leaving a clear patch in the middle, which appears to be a sort of mixing problem. If you run a gel like this the DNA will run in this pattern, so to fix this give the DNA a gentle mix in the well so that the solution is evenly distributed over the large face of the well. Gives much better resoltion when using thick combs, and makes gel extraction much easier!
bob
So you get two distinct bands? Would you suspect one band to be daker than the other? ie: the bottom band should be the darkest b/c most of the liqud does go there??