I have run multiple agarose gels in which I have at least two combs.
I continually see the lower part of the gel visualising much fainter than the top portion.
The last band of my ladder (250 bp) often disappears. It is quite a problem as I often don't see faint bands in this area if I have loaded my PCR products in the lower portion of the gel.
See images attached - look especially at the ladders in the top vs bottom
In the one image you can see the gel itself after a run in the tank, where the loading dye is significantly lighter in the bottom portion than the top - so it's not a problem with the visualising equipment.
I have run the gel for different times (30-60 min) as well as at different voltages (100V vs 120V) and see the same.
The TAE buffer has been changed
I have observed the same with another gel tank.
The intercalating dye, SBYR Safe, has been replaced with a new aliquot.
Other individuals have also experienced the same issue
I use ethidium bromure (BET) to visualize PCR products. This dye migrate in opposite direction of DNA. If my gel run for a too long time the dye risk to be over of small sizes. I supose it is your case.
What you can try: increase agarose concentration at 2% or 3%.
Reduce the time for the migation : 15 min should be enough. You can adjust the voltage.
You can increase your SBYR amount.
Another tip : don't use detergent to clean your gel tank
Are these 2% gels? Was the agarose mixed properly with the dye? Is the gel thickness the same in the top vs bottom section?
@Péter Gyarmati
The gels vary from 0.7-1.0%
The gel is thoroughly swirled with dye a d we use a spirit level to make sure that the gels are evenly poured.
Hi! Ethidium bromide travel in the opposite direction of DNA so the bottom part of the gel get lighter. Nothing to worry. After running stain again and see or run for 2,3 minutes and watch you can see all your products. Good luck!!
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