Troubleshooting: Imaging Agarose Gel with Ethidium Bromide

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Discussion Overview

The discussion revolves around troubleshooting issues related to imaging agarose gels stained with ethidium bromide (EtBr) in a high-throughput PCR-RFLP context. Participants explore factors affecting the visibility of DNA bands, particularly near the edges of large gels, and consider potential solutions and modifications to the experimental setup.

Discussion Character

  • Technical explanation
  • Debate/contested
  • Experimental/applied

Main Points Raised

  • One participant notes difficulty obtaining clear, bright bands for DNA in edge lanes of a large agarose gel, suggesting potential issues with EtBr concentration or UV box lighting.
  • Another participant proposes that the problem may stem from inadequate lighting in the UV box rather than the staining protocol, suggesting the possibility of running smaller gels for better imaging.
  • A participant emphasizes the necessity of running 96 samples simultaneously due to the scale of their population genetics project.
  • Concerns are raised about evaporation during PCR and restriction digestion, with one participant observing that some wells consistently yield faint bands.
  • Suggestions include modifying the light box for better illumination or adjusting exposure times to enhance band visibility.

Areas of Agreement / Disagreement

Participants generally agree that the issue may not be with the EtBr staining protocol but rather with the lighting conditions or potential evaporation. However, there is no consensus on the best approach to resolve the visibility issues, as opinions vary on the necessity of large gels versus smaller ones.

Contextual Notes

Participants mention specific challenges related to gel size, sample volume, and the effects of evaporation, indicating that these factors may influence the outcomes but are not fully resolved in the discussion.

Who May Find This Useful

This discussion may be useful for researchers and practitioners involved in high-throughput gel electrophoresis, particularly those working with agarose gels and ethidium bromide staining in PCR applications.

MatthewHaas
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Hi everyone,

I am running agarose gels (1.5%, 100 V, 100 min) with the product of PCR-RFLP.

My gel has 96 wells...48 on top and 48 on the bottom, so it is a rather large gel...300 ml by volume.

I am having trouble getting clear, bright bands for the DNA in the lanes near the edges. It is very faint. When I shift the gel in the UV box, so the edge is closer to the center, I can see the bands very clearly.

Do I need more EtBr to see this when the gel is in the position I want to photograph? I added 15 ul initially, but reuse the gels (per instruction) and out of fear for overloading the gel, I don't add EtBr each time even though I think it is being degraded when I microwave the gel to get it into a liquid again. When I do, it is on the order of 2-3 ul.
 
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It sounds like the lighting from your UV box isn't adequate for the size of the gels you are running.

I don't think it would be a problem with the EtBr staining itself, if when you shift the edges to center they are clearly visible. Is it possible to run smaller gels and then do your photography and give them a side by side? Or better yet, why are you using the gel size that you are?
 
I need to run 96 samples at a time, otherwise I would consider smaller.
 
I agree with bobze- it sounds like an optics problem, not a problem with the protocol. Not sure how you could easily modify the light box to increase the illumination area (alternatively, putting a central stop on the detector side and increasing the exposure time).
 
I run 96 samples on 100 ml gels. Do you really need the large gels or would you consider running smaller gels?
 
Last edited:
Thanks for the responses everyone.

I run 96 samples at a time because this is a population genetics project and the number of reactions that I am performing numbers in the thousands.

I've done quite a few more gels now, and some have bands clear as day on the edges. I am beginning to think that, where the bands are faint, evaporation occurred in the plates during PCR and/or restriction digestion (even though I use a plastic sticky sheet to prevent this from happening).

Sometimes, it is specific wells that I have trouble with-"repeat offenders", so to speak. For those of you who do 96 samples at a time, how do you make certain nothing evaporates? I am pressing all the edges down, making sure there are no air bubbles trapped inside.
 

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