That first gel looks terrible (faint bands indicating not enough fluorescent dye), but when you poured a fresh gel you got better results. Your samples are barely visible, but present in the first one.

My advice? Pour a fresh gel every time. I bet if you'd soaked the first gel in a buffer with ethidium bromide (or whatever stain you normally use), you would have seen brighter bands.

Is your ladder a pre stained ladder? One possibility if so is that you are not using enough EtBr and electrophoresis pulls it out of the first gel into the running buffer and then you use the same running buffer but now at the second attempt there is EtBr in both gel and buffer and it diffuses into the gel but now there is more dye so possibly a better dna signal.

Hello

This can have different reasons. I suggest some solutions: 1- use fresh gel and buffer for every electrophoresis run. 2-When preparing the gel, add agarose powder to the buffer little by little and mix well so that the agarose gel absorbs the buffer well.3-Allow the gel to boil well when ready.4-Check your pipetting and load more PCR product with load buffer in the gel.

I hope it would be useful

good luck

Electrophoresis is a laboratory technique used to separate molecules based on size and charge. Bands are not typically associated with electrophoresis, as the technique does not typically produce visible bands of separated molecules.

Hello Adriana, it would also be helpful to know what the expacted band size is. Accordingly, did you use a 0.7x, 0.8x, or 1.2x agarose buffer? The lower the concentration of the agarose and higher the voltage used during electrophoresis, the faster is the movement of DNA across the gel, so that could be the reson why there is no bands seen.

Mixing the agarose well in the buffer until the mixture appears transparent, adding the DNA staining dye directly into this mixture, and then cooling the gel mixture on the gel tray until all of the gel turns completely solid (waiting 30-40 minutes at least) could help in the uniform creation of the agarose gel for your purposes.

Also, a well-designed gel run usually has only one band of DNA sample per lane, as agarose gel electrophoresis is only a qualitative confirmation of the presence of your DNA of interest, with the DNA ladder indicative of the approximate size of the DNA concerned. Having multiple bands in the same lane is not considered a good PCR result (as seen in your last lane), so I personally would also check whether the primers used are unique for your amplicon of interest with no danger of forming 3' loops, hairpin structures, etc.

As Hossein Azadinejad and Katie A S Burnette have already mentioned, I believe that your problem is associated with the way you've prepared your gel and running buffer. Be sure to use sterile buffer for your gel and, if you reuse your running buffer, change it now and then, if not every time.

Regarding your post, it would be useful to specify the conditions used, including the amount of loading buffer and sample used in each case. An electronic picture of the gel would also help visualize it better, rather than a computer screen photo.

Good luck with your project!

Hello! What intercalating dye are you using? Do you add it to the agarose gel before it solidifies? In that case, you should remember to check the conditions of the dye. In some cases like SyberSafe, you need to let the agarose cool down before adding it, in others, it has to be hot. Check the datasheet to be sure.

Also, I don't know your lab's budget, but in my experience, for PCR products the running buffer can be re-used 5 to 8 times. In the case of the agarose gel, I try not to reuse it, but I have done it in the past, just melt it carefully not to let it boil and add just a bit more of the dye.

To have clear bands, you need to take into account the thickness of the gel, this one has to be between 0,3 to 0,5 cm. In my case, for a 7x7 gel tray, I use 16 ml of buffer (0,4 cm).

Good luck!

It's possible that there was an error in loading the samples onto the gel the first time, or that the voltage was not applied correctly. Double-check that the samples were properly mixed with the loading buffer and loaded into the wells. You could also try loading a known positive control alongside your samples to make sure that the gel and electrophoresis chamber are functioning properly. Additionally, make sure that the gel is prepared correctly, including the correct percentage of agarose and the appropriate amount of ethidium bromide. It's also possible that there was a problem with the electrophoresis power supply, so you might try using a different power supply or running the gel for a longer period of time.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

i think problem is with the gel preparation and running buffer. properly mix agarose