In recent months, I've been running gels for a fragment around 430 bp on 1.2% agarose gel with a voltage of 120 and a 100 bp DNA ladder, and it has been working without any issues.
After a two-month break, I’ve returned to this routine, but now the runs under the same conditions are turning out as shown in the picture. Does anyone have suggestions on how to improve this? I don’t think it’s an issue with my samples, as even the DNA ladder itself is showing problems.
Hello Lucas Hassib
It seems that your DNA samples and ladder are fine, but there might be a problem in the agarose gel preparation. Are you sure you have used 1X TAE buffer while preparing you gel and run the gel in 1X TAE buffer? I think the gel is made with water or run against water. TAE buffer provides ions that conduct electricity, which is essential for the DNA to migrate through the gel. This poor migration of DNA samples and and "smileys" are typical sign of low buffer capacity.
Best wishes.
I think that 120 volts is too high and is causing the poor separation of the ladder. It may be that previously your power supply had a current limit set for a low current so you were not really running at 120V but while you were away if the current limit was changed or switched off then you run at the full 120v and got this problem. Try resetting the current limit or lower the voltage to 4v/cm (measured between the electrodes)
There is voltage but NO current. Buffer problem is simplest explanation. Alternatively, the buffer reservoir may not be in contact with the gel. Lastly,check the electrodes for continuity. They may have "snapped " while being cleaned. Gremlins showed up while you were away!
I concur with the explanation given above. Have you looked at the concentration of the amplicon and or ladder? Smearing can also result from using too much amplicon and degraded PCR products.
The separation is not good. This is not the separation of 100bp ladder. This could be the buffer, or the voltage. Again check the concentration of the ladder you load or the ladder preparation.
I'd suggest you carefully wash the electrophoresis equipment & make a new batch of buffer. Crusted on salts and/or old buffer can cause this sort of smearing & bunching.
Be very careful to not damage the thin wires in the electrophoresis box, also wash the gel combs, and rise everything in distilled water. Then, try setting up a gel & running just the ladder to see what it looks like before using it for PCR, just to be confident it's working properly.
Good luck!
Running the gel at a higher voltage. Different sizes of gel trays has an ideal range of volts. For example, I found that 5-7 volts may be best per cm length of the gel tray.
Could it be that you added too much of your ladder samples? Otherwise as other answered the problem is the buffer you have used.
I agree with washing all the equipment and making fresh buffer. Get new loading dye too and (if possible) new ladder. Or at least see if you can get an aliquot of fresh ladder from another lab. After 2 months, your reagents could be contaminated (fungus does grow in buffer). Does anyone else in your lab run DNA gels and are they having the same issues or are you the only one who does that sort of work?
If someone new made the buffer (or diluted it from a stock) it could also have been prepared incorrectly. I had a summer intern who wanted to be helpful and kept on refilling bottles with just tap water. We got a lot of weird results until I figured that part out!
Intenta disminuir el voltaje, 120v es mucho para un gel al 1.2%, si tu cámara de electroforesis es pequeña, estas se pueden sobrecalentar y dar bandas con irregularidades, tu ladder no se abre justamente porque está migrando muy rápido, además si ese ladder lo estas usando por mucho tiempo puede también que este algo degradado por congelar y descongelar, utiliza una nueva alícuota para probar con menos voltaje, saludos! y disculpa por escribir en español!
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