Hi all,
can you give me some opinions/ suggestions on why my agarose gel comes out like this on the bottom after the run? It always happens to me with the larger gels, never with the small ones. I usually run the gels at 60 V for 1hour and 30 mins-2hours.
Thanks
if you are saying that if you loaded the lower samples at the top of the gel then they would run clean and have bands then i think that your gel tray is deforming under the heat of the agarose and the comb is touching the gel tray. Then there is some leakage of sample because the well has no bottom after you remove the comb. I suggest 2 things.
wait until your gel is a bit cooler before pouring the gel
and/or raise the comb a little with some form of spacer ( folded paper perhaps) to keep the comb higher off the base of the gel tray.
One reason I say this is that the size standards are running better than the samples which makes sense as the edges of the gel are less affected by tray bending as they are held in place by the edge pieces of the perspex tray. The middle of the gel tray bends the most.
if you are saying that if you loaded the lower samples at the top of the gel then they would run clean and have bands then i think that your gel tray is deforming under the heat of the agarose and the comb is touching the gel tray. Then there is some leakage of sample because the well has no bottom after you remove the comb. I suggest 2 things.
wait until your gel is a bit cooler before pouring the gel
and/or raise the comb a little with some form of spacer ( folded paper perhaps) to keep the comb higher off the base of the gel tray.
One reason I say this is that the size standards are running better than the samples which makes sense as the edges of the gel are less affected by tray bending as they are held in place by the edge pieces of the perspex tray. The middle of the gel tray bends the most.
As an after thought if you always load the lower wells first and are very slow in adding the samples then you may get diffusion of the samples that you loaded first which could smear the bands. If you think this might be the case then either load quicker or load one row then run it into the gel for 5 minutes then load the second row
Ok, great!!I will try. Thank you very much Paul for these helpful suggestions
On Seeing the gel, i feel that something is wrong with the comb used for the bottom wells, so that well is not formed properly and the samples has run like smears. But the Ladder at the extreme right of bottom gel has run properly. Or may be, as you are running the gel for longer duration, heat generated would have deformed the gel and hence the samples are not run proper.
Suggestions:
Try different comb while casting the gel.
Increase the voltage to 80 V and run for 1 hour. If the gel is 2% or more, increasing the voltage will not affect the quality of gel.
Time taken to load the samples also matters, quick loading would reduce the problem of diffusion of samples in the gel.
Since you get clear bands on some of the wells and the ladder seems to have no problem, it might also have something to do with the quality of the samples or their concentration. I suppose you load the same quantity in each well. I also see that in some wells the sample has not run, so probably it's too concentrated. Alternatively you could try different agarose concentrations..
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