5 Ways to Get Better GelRed Results*

Switching from ethidium bromide to GelRed^TM offers a safer and more sensitive option for visualizing DNA in agarose gels. However, some researchers report inconsistent or faint bands when using GelRed^TM, often due to the way it’s applied.

To get the best results when using GelRed^TM, perform post-staining, avoid adding GelRed^TM in the running buffer, and adjust your dye concentration. Along with these, there are other approaches to optimize your GelRed^TM results.

In this article, we’ll take you through some standard approaches when using GelRed^TM that should help with your results. Then we’ll go into some special tactics that might be fitting when you’ve tried everything else or are encountering a special situation.

Article Table of Contents

1.Post-staining with GelRed^TM instead of precast staining

Post-staining with GelRed^TM

When it is ok to pre-cast gels with GelRed^TM

How to pre-cast your gel using GelRed^TM

2.Don’t add GelRed^TM to running buffer

3.Adjust your agarose concentration based on your DNA size

4. Increase stain concentration or staining time – When and How

5. Warm your post stain solution

1.Post-staining with GelRed^TM instead of precast staining

One of the most effective ways to improve results is by using a post-staining protocol rather than mixing the dye directly into the molten gel.

Just for clarification, post-staining in electrophoresis is where you would stain your gel with a nucleic acid dye after the gel ran (after electrophoresis).

Unlike precast staining, post-staining avoids risks like thermal degradation and inconsistent dye distribution.

GelRed^TM is more heat sensitive. So, GelRed^TM shouldn’t be added to molten agarose because the high heat may reduce its staining efficiency, lead to weaker bands, and that can lead to wasting good product.

Another issue with pre-cast staining when using GelRed^TM is that you may run into inconsistent dye distribution. Inadequate mixing after cooling leads to uneven staining, streaks, even blotchy patterns on the gel.

Post-staining with GelRed^TM improves band sharpness and resolution and gives you more uniformed results.

Post-staining with GelRed^TM

1. Run your gel as normal, without GelRed^TM in the gel or running buffer.
2. Prepare a 3X staining solution by diluting GelRed^TM 10,000X stock to a 3X final concentration. For example, add 15 µL of GelRed^TM (10,000X) and 1 mL of 5M NaCl to 49 mL H₂O.
3. Place the gel in the solution and incubate for 30–60 minutes at room temperature with gentle rocking or shaking.
4. Visualize the gel using an ethidium bromide filter.

Post-staining not only delivers clearer results but also simplifies cleanup and prolongs the life of staining solutions through reuse.

When it is ok to pre-cast gels with GelRed^TM

While GelRed^TM is better added during a post-stain procedure, pre-casting with GelRed^TM still commonly occurs in the lab. And there are times when pre-casting can be appropriate.

For instance, if your results don’t need to be super precise, pre-casting is a convenient option that can still work.

Or, if you’re doing a quick check of your PCR or digestion, you could pre-cast your gel with GelRed^TM.

How to pre-cast your gel using GelRed^TM

Start by preparing your agarose as you usually would, dissolving TAE or TBE using your lab microwave. Wait until your melted gel has cooled to ~50–60°C.

Hotter liquid agarose can degrade your GelRed^TM. Wait at least 10 minutes after boiling. Or, to be really sure, my mentor had us wait until the glass wasn’t too hot to touch. If we were comfortable picking up the flask, then it was safe to add stain and cast. So once it’s cooled enough, add your GelRed^TM.

Swirl your flask to evenly distribute your GelRed^TM. Be careful to avoid getting bubbles. Once it’s mixed, pour your gel into the tray and insert your comb, allowing your gel to solidify before using.

Now, with all of that said, if you’re looking for publication-quality gels or working with very small fragments, you definitely want to stick with post-staining instead.

2.Don’t add GelRed^TM to running buffer

When transitioning from ethidium bromide to GelRed^TM, another thing that happens is researchers will add GelRed^TM to the running buffer just like when using ethidium bromide.

Unfortunately, while going this route feels intuitive, it can hurt your results.

GelRed^TM is more chemically complex than ethidium bromide and it strongly binds to DNA. When it’s added to the running buffer, it floods the gel with free dye, which leads to higher background fluorescence, increased noise, and can lead to dye diffusion in the gel.

So, what do you do instead? Run your gel as you normally would. Then after it has run, put your gel in a staining solution.

• Run the gel without GelRed^TM in the gel or running buffer.
  Your gel should be cast and run using TAE or TBE buffer.
• Post-stain the gel.
  After electrophoresis, place the gel in a container and add your staining solution of 3X GelRed^TM.
• Incubate with gentle rocking for 30–60 minutes at room temperature.
• Image the gel using an ethidium bromide filter. Optimize your settings if necessary.

You can use your staining solution 2-3 times. Be sure to store it at room temperature, and keep it protected from light.

3.Adjust your agarose concentration based on your DNA size

If you’ve been using GelRed^TM and are sometimes getting compressed or smeared bands, or your bands are hard to read, it might have to do with your agarose concentration.

Choosing the right agarose concentration is a standard practice when doing electrophoresis, but having it right becomes even more important when using GelRed^TM because it is larger than ethidium bromide and is a stronger intercalating dye.

These properties can change the way DNA migrates through your gel. If your gel concentration isn’t optimized for your DNA fragment, you might run into resolution issues.

You can also start with a set percentage and adjust up or down from there depending on how your fragments resolve.

This advice isn’t just for using GelRed^TM. It applies to any gel you’re setting up. However, the effects are amplified when using GelRed^TM due to its larger molecular weight and strong intercalation.

4. Increase stain concentration or staining time – When and How

Another reason you man run into issues or experience faint bands when using GelRed^TM could come down to your stain concentration or the staining time. Simply adjusting either of these can help you optimize your results, especially if you’ve been used to working with EtBr protocols.

When working with GelRed^TM, you want your working concentration to be 3X from a 10,000X stock.

If 1X -3X isn’t getting you the results you’re hoping for, experiment with concentrations up to 5X. This can be really important if you’re reusing your stain solution.

Aside from ensuring you’re using the right concentration, another thing you can try is to increase your staining time to about 60 – 90 minutes. This is going to be a good strategy to try when you have a thicker gel or you’re working in a colder lab.

5. Warm your post stain solution

Something that can be overlooked when staining your gel is temperature. Because GelRed^TM has a higher molecular weight compared to EtBr, it diffuses slower, especially in cold or thick gels. This is where warming your stain can improve your results.

Warming your staining solution is going to be a great option to try when you’re in colder environments. This can also sometimes help if you need to reduce your staining time but don’t want to increase your staining concentration anymore.

When warming your staining solution, keep your target temperature between 30°C - 37°C, being sure not to exceed 37°C. Warm your solution for about 10 - 15 minutes before using it. Then, while staining, gently rock to make sure you get even staining throughout.

Warming your post-stain solution is not going to be needed every single time. In a standard lab where temperatures are usually between 20°C - 25°C, you may not need to do this, especially if you have a thinner gel. But if your lab is kept cooler or when you need to troubleshoot your results, warming your gel takes little effort and can improve your results.

When it comes to optimizing GelRed^TM, a lot of this has to do with understanding that it works a little differently compared to EtBr, which means you’ll have to break some longstanding practices if you’re switching.

But the good news is, once you get it down, and really understand the settings and setup, you’re going to get the results you’ve been looking for along with the enhanced safety that comes with GelRed^TM.

*Disclosure: This article was researched with some assistance from AI. The article was written by Katharine Martin and was edited by Simon Currie.