Thanks Catarina for your answer.

We always add the intercalating dye to the sample rather than staining the gel after the electrphoresis (it is a practice coming from the times we used ethidium bromide to reduce the amount of a toxic chemicalk). But anyway the point is that GelRed sometimes changes the migration and sometimes does not, and this behavoiur can be observed with the same DNA fragment obtained in two different PCR reactions. I wonder whether GelRed, besides binding nucleic acids, is also able to simultaneously bind other molecules that  are present in the sample (e.g. proteins or carbohydrates coming form the original DNA prep).

GelRed's size is comparatively heavier than EtBr, thus effects the migration speed in gel. Though we add this stain by pre-staining or along with sample into wells, it shows same effect most of the times. 

What catarina told is logical and really worthy.. post staining is better than prestaining or adding stain to sample while loading. 

I always feel prestaining reduces the time for gel electrophoresis. Safe handling and safe disposal of any stain is priority as per we've been taught. 

This gelred may also bind to other than nucleic acids like proteins but it is a good phenomenon you'll see on gel to roughly estimate the visible impurity of DNA sample. This is what i really believe in.

As far I can say from my experience using SybrGold in the sample, it decreases the mobility of DNA, which is  proportional to the amount of DNA molecules in each sample.  So to say, more DNA less mobility. 

Whether this applies to the GelRed as well, I do not know, however similar dyes from different companies I tested behaved the same way as SybrGold. 

Luis,

I use GelGreen and have had this same problem when precasting my gels. I have found that cutting the GelGreen concentration in half (0.5X instead of 1X) seemed to work or loading less of the PCR product into the pre-casted wells --- either of those options didn't affect the mobility of the PCR product. I wouldn't recommend adding the stain directly to your PCR product just for the fact that the mobility may be affected, as you and the others have said. You could always do the post-run staining as the others have said.

Cheers,

Chris

Thank you all for the advices. It seems that there is no other good option but to post-run stainning of the gel.

Hi luis

I had this problem before, the other problem that I encountered was the fluctuated band which appears on the gel, after many attempt, we notified that the balance of gel red, water and loading buffer and also PCR product, is so important. It was about some times ago and I can’t remember the exact amount of them. But I suggest you using SYBER GREEN or EtBr, they are much better.

Good luck

The other thing that I forget; I used Gel Red to post-run staining of the gel, but when I watch that with UV transluminator it had bright and white background, and the picture quality wasn’t good at all.

You can try methyl green staining, although you would need a red/far-red illumination device (G:box or similar). MG is a small molecule, it is cheap and safer than EtBr.

Best of luck,

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Hi Luis,

I have noticed the same effect with GelRed as sometimes migration looks unexpected (usually as if the bands were of a higher MW).

Information about this effect are here:

http://biotium.com/wp-content/uploads/2013/07/GelRed-and-GelGreen-FAQ.pdf

It looks that one way is to reduce the concentration of the dye and the concentration of the DNA to 50-200ng per lane (measured by Qubit or Nanodrop? This is not reported even if DNA concentration for DNA non-ethanol precipitated (i.e. miniprep) is very different using Qubit or Nanodrop (the former being much more accurate to me).

It is also worth noting that some suppliers sell loading dyes containing SDS (about 0.02%) to avoid "band-shift" effects due to restriction enzymes bound to the DNA (DNA migrates at higher position as still bound to restriction enzymes, frequently engineered in the DNA binding domains).

As suggested here (https://www.neb.com/products/b7024-gel-loading-dye-purple-6x) some of these dyes may contain more SDS and this may interfere with GelRed in a way not specified (smearing of bands?).

Personally I use happily GelRed as it is sensitive and safe but it is important to control the above effect, and I am trying these tips actually.

Max.

Wow, I wish I would have found this thread awhile back, I could have saved myself a lot of time! I have been trying to figure out strange migration patterns with our 1 kB ladder on an agarose gel, first thinking what is wrong with our buffer, our power supply, etc. when the whole time it was adding GelRed directly to the gel. Post staining completely solved the problem.

I sometimes have seen the same behavior.

A much longer migration appeared to sort out the problem at least in one case.

Not sure why this happened, but it may be something affecting more bigger fragments that get resolved only when they migrate extensively.

I have seen my band occupying different positions compared to the ladder until the expected size was reached after a certain point.

Again it is not always happening and this is the confusing aspect.

Max.

true Massimiliano Zampini  sir

I´ve noticed the same. When using GelRed in the loading buffer to prepare both standard and samples, even the same PCR product run on the same reutilized gel (during a cloning procedure - first to check size, then to control undigested product) migrated differently. That never occurred to me in the past with EtBr staining. When I switched to post-run staining (30 min 3x GelRed in MQ water, 50 mL for minigels) everything showed on the right size. Even for qPCR, sometimes I obtained double bands when using GelRed in the loading buffer, despite a single product was obtained (dissociation curve analysis). I definitely think that, when precise size determinations is required, post-run staining is the best when dealing with GelRed stain.

Yes, GelRed can affect the migration of DNA fragments. All DNA binding dyes do so, but it is more frequently observed with GelRed because it is a larger dye. The migration effect is dependent on DNA loading, higher DNA loading amounts cause more pronounced migration effects. Usually reducing the amount of DNA loaded fixes the problem, but if very precise sizing of fragments is required, we recommend using the post-staining protocol.

Check out our FAQ for tips on troubleshooting GelRed: https://biotium.com/support/faqs/faq-gelred-and-gelgreen-nucleic-acid-gel-stains/

Yes, Gel red does affect DNA migration, supercoiled DNA runs at higher  than normal. The migration is also affected by concentration of your DNA sample with gel red. If you run two different concentration of sample with gel red, you see them clearly one with lower concentration runs slightly higher than other.

We've had the same issues whereby a PCR product can appear as much as 500 bp longer than it is. It's inconsistent as well, but when it does affect the speed, it always slows the migration making the products appear larger than they are. It's exceedingly annoying, and as mentioned, post-staining is the only sure solution save using EtBr.

Dear Fellow researchers, thanks for the information about EtBr and running DNA samples. I was looking for information how supercoiled DNA acts in EtBr and GelGreen/GelRed? Anybody have experience?

I am using GelGreen and GelRed for 7-8 years now without any problem, I have never thought to put it into the sample or gel, since it is clearly written that it requires post staining (which is like 5-10 min). Very honestly, why people do it?

Dear Fellow researchers, thanks for the information about EtBr and running DNA samples. I was looking for information how supercoiled DNA acts in EtBr and GelGreen/GelRed? Anybody have experience?

I am using GelGreen and GelRed for 7-8 years now without any problem, I have never thought to put it into the sample or gel, since it is clearly written that it requires post staining (which is like 5-10 min). Very honestly, why people do it?

Yes it dose ad ruined almost 6 month of my work before I noticed about this effect. Cloning of RNAi construct just gone away. I never could trust any of my positive clones.

Gelred migration effects are also accentuated by gel:DNA ratios. You can look up their new Prestain Plus loading buffer's insert which shows dramatic effect of the older iteration ( https://biotium.com/wp-content/uploads/2018/11/PI-41011.pdf )

I have noticed that, because dye doesnt migrate, later (higher kd) bands will be fuzzy if you dont add the recommended conc. (like how Stacey Wood Scott used 0.5x) or if you add too much DNA.

Ultimately I feel that GelRed is not better than EtBr under all circumstances. Your requirements will make the choice.

I was glad to find this thread! GelRed really messed up my PCR amplicon migration relative to the size standard. Post-staining the gel sorted out the issue.

pls inform me if the problem here is in the used red safe. im using the red safe of vivantis co, Malysia. using 5 microliter per 100 ml of agarose. pleease look for the ladder and also the samples. please help. thanks

The problem you have seems different from what I was talking about. It seems that you have a general migration problem that could be due to either the electrophoresis or sample buffer.

The problem with gel red or equivalent dyes is that sometimes DNA fragments do migrate slower in the gel than expected according to their sizes