We prepared PCR products and ran a gel on a new SynGene gel doc system with Sybrsafe dye and compared the result with a gel with ethidium bromide. In one case the bands were located at ~5.5kb (ethidium bromide), and in the other they were at ~6kb. Has anyone else noticed this? Explanations?
I had the same problem. You can solve it staining your gel after electrophoresis.
Are you using the same ladder in both cases? I haven't used Sybrsafe myself but if the system makes a band looks like the wrong size systematically, I thought it would have been caught long ago. Do you see the same with smaller products using the same ladders too?
Yes, same MWt ladder in both cases. We just got the new system installed, so we are just getting into using it. Seems strange. Maybe we made some strange error, but not clear what.
Thank you both for your answers. I am troubled by this result, especially given that others have the same finding. We'll look into Midori green. Again, thanks, Bob
I had the same problem. You can solve it staining your gel after electrophoresis.
This is normally occurring in most stains, because either you add opposite charge to DNA (in case of ethidium bromide which is +charged) or change bulkiness of DNA in SybrGreen. Both are sensitive to the DNA/stain ratio. You can stain DNA with ethidium that will not move anymore. The best solution if you need to determine exact position of bands is to stain after electrophoresis as suggested by Kirill.
Hi Robert,
Just to chime in, we use Gel Red, and I observed the same effect. A PCR product of ~660bp was on par with a 750bp band of the ladder. It cost us a lot of time, since we suspected the product was non-specific, and tried to optimize the PCR. When I ran the same amplicon on EtBr, it turned out it was exactly the right size. It was also observed by another lab member, but is seems that the relative "size distortion" gets worse the longer your piece of DNA is. We tried the recommended post-staining, but the bands on gels came out really faint, and it uses a lot more stain.
1. Those safer DNA dye, including GelRed™ and GelGreen™, can affect the migration of DNA fragments on the gel. This has also been discussed on RG at: [ https://www.researchgate.net/post/Does_anybody_know_why_GelRed_modifies_the_migration_of_DNA_fragments_in_agarose_gel_electrophoresis ]
2. According to the explanations of the company (Biotium) producing those dyes is:
".....because GelRed™ and GelGreen™ are high affinity dyes designed to be larger dyes to improve their safety, they can affect the migration of DNA in precast gels. Some samples, such as restriction digested DNA may migrate abnormally in GelRed™ or GelGreen™ precast gels....[ https://biotium.com/support/faqs/faq-gelred-and-gelgreen-nucleic-acid-gel-stains/ ].
3. Therefore, the best way is to stain the gel after run.
As said before both SybrGreen and GelRed are dyes designed to bind the DNA without intercalating within the Watson-Crick pairs, to improve their safety. Compared to EtBr they are also larger molecules minimizing, as possible, their ability to pass gloves and skin. By working in this way they can affect the migration of the DNA, specially large DNA fragment. In my hands GelRed greatly distorced bands larger than 1000 bp, so I was used to stain the gel after run. We have switched to SybrSafe from MolecularProbe and we have solved the problem.
Okay, on further thinking about this issue, I am puzzled, again. If the molecular wt ladder is run at the same time as the sample DNA, the ladder fragments should bind the dyes and be affected in the same way as the sample DNA. So, regardless of whether the electrophoresis is run with dye present or dye is added after electrophoresis, shouldn't apparent molecular weight of the DNA sample still read correctly?
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