As far I can tell, two major factors could be responsible, the storage condition as well as the running buffer. How do you store the ladder and are you using a fresh buffer or an old one ? And finally, it might be silly, but are you loading the same volume because it appears that the latter ladder have lower concentration of DNA? How about the EtBr concentration ?

You pipetted the sample in an angle, and not straight in the slot. Or there was a little nick in the slot when you put the sample in there.

use fresh running buffer and check the running buffer temperature while your ladder running. The nucleic acids are degrade slowly under warm buffer condition.

It looks to me like you have something growing in the ladder mixture and degrading the dna. Is this standard stored at room temperature,cold or frozen and re thawed each time

The possible melting of agarose was not complete, cold agarose was used when pouring the gel.

The possible in the loading buffer a lot of glycerin or ficoll.

DNA in the ladder did not degrade. The problems are not with the ladder, but with the electrophoresis, agarose or buffer.

Poor quality of the agarose gel, especially at the well.

Hi,

When I have bad ladder resolution I review the pH of the electroforesis buffer and review the agarose concentration. Also the date of buffer preparation and the conditions of storage are very important. The salts of the buffer, the voltage and the time you spend in the electroforesis affect the quality of the running. I recommend SB Buffer for electroforesis at high voltage.

Try to run another electroforesis with another ladder and the same agarose and buffer just for check if the ladder is the problem.

Thanks for your answers.

These are very helpful to my research.