It looks like the samples are running in a high salt sample (pcr or sample) buffer. When you say old cells new primer was the dna from the old cells made fresh or is it old stored dna that should run cleanly. Is there anywhere in your sample preparation that too much salt could be occurring?, Especially anything that has changed in the sample preparation since that worked well, This could effect both the pcr and the running of the pcr product. The middle size ladder does seem to be running oddly at the lower sizes close to the samples which also suggests too much salt in the sample wells is having an effect on the ladder as well as making the samples run unevenly

Paul Rutland Hi! thanks for your answer. By old cells I meant cells I had collected a week or so earlier, were stored at 4C and was re-running to see if there was a difference between the newly collected cells and the older cells. I realize I should also have a positive control to see if it's my primers or the gel or something else that is happening. As per the salt, I had thought it was perhaps the buffer in the rig because we keep the same buffer in the rig for multiple days but some of the water can evaporate making the buffer have a higher concentration than it should. I replaced the buffer we use in the running rig for fresh buffer that should be of the right concentration. I'm not sure if that is what you meant by that, sorry if I misunderstood. Also worth noting that other people in my lab have run their gels in the same buffer and are having no problems so i'm assuming it could be my samples themselves that may be the issue.

Maybe a basic question, but are the gels prepared using the same buffer that is added into the rig? Also, by letting the gel set for 1 hour, I'm assuming you mean to solidify? (As opposed to letting the solidified gel sit in the rig for 1 hour.)

Gel buffer can be reused a few times but eventually nucleases grow and the nature of the buffer changes. It is best to have the same buffer concentration in the gel and the tank and if you are reusing the same running buffer then mix it well before use as it becomes acidic at one end and basic at the other end and results are variable. You may be getting worse results that the others because your bands are smaller so reach the ends of the gel quicker and are more effected by pH and salt effects. You could also try running at half the voltage to avoid heating effects and you may find that the bands look better if they do not run so far and the results will still look good even if run less far