Hi,
I run an agarose gel for 4 hours and I noticed that my bands after that time were more fainted. Gel Red has been added to the gel during the preparation and the DNA loading buffer was added to the samples before the run. Can it happen that the dye or the gel red get fainted as the run goes on? If yes, is it better to stain the gel after the run when I run it for many hours?
Thanks
Running an agarose gel for as long as 4 hours can lead to several issues. I am not sure what your running conditions were, but long runs can generate lot of heat causing the agarose to melt. It can affect your band appearance. If heat is not an issue, you can stain the gel after the run.
I wonder why you did run your agarose gel for 4 hours. Usually 30 minutes are sufficient for mini-gels under standard conditions. Do you see the fainting mostly in the lower end of the gel? This happens will all gels that the small molecular bands and loading buffer dye bands appear fuzzy and the longer you run it the more fuzzy. You can see it here on the pics from the GelRed website too: https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/.
Now on the other hand if the run finished but can't image the gel immediately I leave the gel in the chamber with a very low voltage. Without any voltage all bands will start to diffuse and faint.
Dear Silvia
- In short yes if you run for prolonged periods - more than 1 hour - you will see a reduction in staining particularly of smaller bands
- From what I can ascertain gel red unlike ethidium bromide doesn't migrate up the gel away from DNA fragments migrating down the gel during prolonged electrophoresis
- In effect bands are therefore detained by prolonged (> 1hour) electrophoresis in gels containing ethidium bromide
- I use Gel red and have never seen that affect as markedly
- However, I have post stained gels by adding gel red to the running buffer following electrophoresis for more than 1 hour, and in my experience this has led to more uniform band staining
- In the past it was recommended for prolonged electrophoresis times with ethidium bromide to add to both gel and running buffer and certainely this cannot harm with gel red
- Or alternatively, as I have done with gel red, add to the running buffer post electrophoresis and allow to stain for ~ 20 min ( I would still add to the gel though)
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