Hi all,
I attached a picture of an agarose gel because I can't obtain straight bands, especially when I run my ladder (first and last well). They form a sort of wave. I run DNA samples on 1% agarose gel.
Can anyone tell me on what does it depend?
Thanks
Hi there,
Make sure the sample is evenly distributed in the well before applying current. To do so deliver the liquid as slow as possible and as deep as possible into the well. an other way of doing it is to increase the volume of the sample.
I think the waves you see in your bands are due to the voltage you are using to run your samples. In general, a 1% agarose composition should be fine for your samples, although there isn't much detail on them to make an accurate comment. What is the overall volume of your gel? Do you make it in your buffer or in water?
I would also consider looking at a similar question on ResearchGate: https://www.researchgate.net/post/Why_do_DNA_band_in_agarose_gel_start_to_become_curved_and_wavy_as_it_travels_down_the_gel
Yes I am using the gel red during the gel preparation. I am preparing a 1% agarose gel in 1X TAE buffer. I load 2 ul in the well and I run the gel for 2 hours at 50V
This has nothing to do with the stain, and there is nothing wrong with the DNA loading. Two possible reasons for this effect: 1. You waited to long between loading and running, so adsorption/diffusion into the well separators has occurred and the edges of the lane are then retarded compared to the centre of the lane because part of the sample is running through the gel separators (but this usually causes vertical streaks at the lane edges), 2. When you pull the comb (maybe too fast, or too early) you have pulled up the centre of the well bottom, so the "floor" of the well bulges like an inverted "U" (not as exaggerated, of course). If you load a small volume, it will occupy mainly the trenches on either side of the distorted well bottom (because the dense sample sinks into them), so there will be progressively more sample towards the edges of the lane than in the middle—and also the running at the edges will be somewhat retarded because there is more effect from the sample buffer vs. the running buffer than in the middle. Judging from the shape of the bands, I would say #2 is most likely. The best solution: it is always best to use a volume of sample that fills the well to about 80% of available volume (i.e. use more volume, as has already been suggested). Also, take care in pulling the comb and make sure the gel is firmly set when you do it, and try to load and run without delay. Running the gel excessively fast can also cause distortion, but this is usually overall, rather than well-by-well.
@silvia, I'm 90% sure it is the gel red. For instance, your DNA ladders are super smiley as compared to your samples. This is proof that too much DNA interaction with GelRed creates this effect. See attached pic. I often get similar gels when using a GelRed-like product. Doesn't do it with another product.
Thanks for the follow-up, Simon. That seems a little more convincing, but an example of straight bands on a duplicate gel (with whatever condition you think works) would have been even better. I am still perplexed as to the theoretical basis for the band shape as a result of a uniformly distributed DNA/dye concentration. I hope you post what works in the end for you, Silvia.
All above suggestions are good and worth to try. I suspect that the edges of the teeth of the comb need to be smooth. Also one can apply little silicon grease to prevent pulling of the gel and distorting its surface.
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