hi

may be your DNA concentration is low

it looks like a very strong band at 100 bp to me. A bit diffuse but small bands often spread due to the heat of the gel. Run it at half your run voltage if you need a tighter band but assuming that it is the right size then it looks fine to me

DNA concentration doesn't seem to be the issue here. There are a few things you can try.

1. Try running the gel at a lower voltage if you have been running it at high. More often than not, this solves the issue. However, if you have run your gel at the same voltage for quite a while now, try the following.

2. Change the water you use to make TAE and running buffers. Make fresh stocks. Often, the water quality might go down without anyone noticing leading to such a profile.

But yes, the first suggestion should do the job.

Dear Friend,

It may seem sarcastic but note worthy that sometimes there can be problem with the camera lens and zoom function in the Gel Doc system. First check that. There seems absolutely no problem with the DNA concentration and PCR. Also try running the products at 1.2 or 1.5% Agarose gel with lower voltage to avoid heating of the gel. Another important thing is the recommended thickness of a gel is 3-5 mm. If the gel is more thicker then the bands may appear fuzzy.

Hey,

Thank you for all the answers so far.

My thought was that it might had to do with the gel as well. It was an EtBr gel which was made 5 days before running this gel. I was told I could store in RT but I don't know whether that is OK. I was thinking on making a new gel to try alongside the older one in case that had to do with anything.

Anirban, the gel was run on 100V/45mins. I could try with 50V perhaps.

It is a SELEX protocol so I'm not expecting a very high concentration this early on either.

Anirudha, sadly I don't think it's the camera. I've scanned PAGE gels just fine before and it also looked faded under a UV table.

Hello

Did you make the gel and store it in a closed container at 4 degree's celcius totally submerged in TAE? If you did that's fine, however not adding extra amount of (about 3-6 microlitres) of Etbr into the TAE buffer where you put your agarose gel could be also a possible source of problem because due to long storage time, your EtBr on agarose gel could have leached out of the gel. As such intercalation would not be effective. Also the faint band may be due to low concentration of DNA in your sample.

I run two gels - one I made 8 days ago and one I made yesterday - but I didn't see any difference between the bands unfortunately. The gel I had it stored in a plastic bag in room temperature with no buffer

You said: " It was an EtBr gel"

Did you use EtBr directly in the agarose gel?

Did you add EtBr to the electrophoresis tank too?

I think, you'd better stain the DNA after electrophoresis. Use 1.5% agarose gel instead of 2%, I had problems with high concentration agarose gels and finally, use lower voltage.

Thanks for the suggestion Aref.

To answer your question I only add EtBr in the gel when I'm casting it but not in the tank.

But after 2 weeks of trying different possibilities I have found the best conditions for my DNA fragment. This is the same DNA sample as in the first picture and you can see it looks so much better now.