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What are the optimal staining conditions when using Gel Red? So far, since we have started using it, the gels ran in our lab have been of very poor quality. The bands are very blurred and often indistinguishable. We have tried both pre- and post-staining, together with varying concentrations of Gel Red.

Any advice about how to fix this problem?

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The gelred tag seemed a bit too specific... –  user132 Dec 15 '11 at 15:53
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Are you sure it's the staining, and not the gels themselves that are the problem? –  Mad Scientist Dec 15 '11 at 17:58
    
What wavelength and filter set are you using for visualizing your gels? –  Mac Cowell Dec 16 '11 at 3:30

2 Answers 2

I have had success with 1.5% agarose gels in TBE pre-stained with gelred. I use a 10,000x stock solution of gelred sold by http://biotium.com. I visualize the gel on an old fotodyne UV transilluminator.

After melting agarose in the appropriate volume of TBE in a microwave, I let it cool to about 50 C (I can touch the flask with the back of my hand without snatching it away) and then add the appropriate volume of stock gelred.

For a 30 mL 1.5% agarose gel:

  1. mix 0.45g agarose + 30 mL 1x TBE
  2. microwave until agarose solution boils and clarifies
  3. let cool to around 50 C
  4. add 3 uL gelred
  5. swirl the mixture
  6. cast the gel (don't forget comb)

This typically works for me. I run the gel in a 1x TBE buffer at around 120 volts.

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i've also had the best resolution with pre-stained gels. Even so, its better to take long time exposures of the gels on UV sometimes. Hi Mac! –  shigeta Oct 4 '13 at 15:01

I don't think you should need to vary the concentration of the GelRed. Mine came with instructions for the exact concentration and how to dilute. Optionally salt can be added, which I did, and it has worked for me.

I have only done this with Post-staining. 1-2.5% agarose with TAE. Used a 10,000x solution from Phenix research. The gelred stock and working solution needs to kept in the dark. I am assuming you have a transilluminator and (optional) filters to visual though. GelRed has similar excitation and emission [wavelength] properties as EtBr, so a standard transilluminator and filter should work without any changes. It will not be visible with the naked eye.

That being said there are a ton of things that can go wrong in casting the gel, eletrophoresis, staining, and visualization. If bands are visible but just blurry I was suspect there is more of a problem with either the gel creation or eletrophoresis though. Make sure agarose is completely clear and cooled down a little before pouring. Otherwise voltage is one of the main things I have noticed that can have significant affects on how sharp or blurry bands appear. You might try different % gels and different voltages. There are formulas that can help you estimate the best value for each of those.

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