Do the bands overlap with the migrated loading dye? If you have a high concentration of loading dye in the samples, it can occlude the bands underneath it. I've always extensively diluted common DNA loading dyes with 50% glycerol so that this effect is negligible.
If this isn't the case, it could even be the UV lamp source; the top part of your gel may be directly underneath the lamp while the bottom is not. I've had this problem with certain imagers in the past.
The bands that move furthest in agarose gels are the smallest size bands. Becuuse they are small molecules they are most effected by thermal diffusion....the hot molecules of buffer ions hit the molecule and knock it randomly in all directions. This is thermal diffusion and it makes the bands spread in all directions so they look diffuse and weak. They also look weak because the ethidium bromide or other dna stain binds between the dna strands in a manner related to the length of the molecule so small dna binds less ETBR and fluoresces less than large dna.
To get sharper clearer bans cool the gel and buffer and run it in a cold room and run at a low voltage (so less heating of the gel and buffer) and also use a higher percentage agarose gel. These 3 things will give much clearer bands
Thank you Aaron P Landry sir and Paul Rutland sir for your valuable response.
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