I have been attempting asymmetric PCR Lately and have noticed that some of my bands on the gel are showing up in an orange color vs the typical greenish color. Could Gelstar dye bind ssDNA in a different manor then it does dsDNA resulting in a slightly different florescent emission?
I have read that ssDNA travels ~10% faster than dsDNA. This suggests that the orange bands are ssDNA, but I want to make sure. the green bands I am seeing correspond in size to standard dsDNA PCR lanes with these same primers.
Could this perhaps be some other contaminate or perhaps the DNA is taking on a unique confirmation?
If anyone has experience with this phenomenon, I would really appreciate your help.
*I have attached the gel picture for reference
The GelStar stain detects both double-stranded (ds) and single-stranded (ss) DNA. It also detects oligonucleotides and RNA (native, glyoxalated and formaldehyde-denatured RNA).
See also the paper:
White , H.W.; Vartak ,N.B.; Burland, T.G.; Curtis, F.P.; Kusukawa, N. (1999) GelStar nucleic acid gel stain: high sensitivity detection in gels. BioTechniques 26, 984-988.
The emission wavelength of ssDNA is somewhat different from dsDNA. However, it should be mentioned that the color is also influenced by pH and chemicals that interact with the stain/nucleic acid complex. The total emission wavelength depends on the filters that you use.
Hi David, I have similar experience. The emission of single stranded DNA labeled by Gelstar is shifted to red. I often see it when I was amplifying cDNA. I think it is difficult to estimate the speed of ssDNA migration since it depends on dsDNA conformation and on condition like running buffer composition. If you want to measure the size of ssDNA fragments it is better to run PAGE gel under denaturing condition, the speed of migration will be same for both ss and dsDNA.
The GelStar stain detects both double-stranded (ds) and single-stranded (ss) DNA. It also detects oligonucleotides and RNA (native, glyoxalated and formaldehyde-denatured RNA).
See also the paper:
White , H.W.; Vartak ,N.B.; Burland, T.G.; Curtis, F.P.; Kusukawa, N. (1999) GelStar nucleic acid gel stain: high sensitivity detection in gels. BioTechniques 26, 984-988.
The emission wavelength of ssDNA is somewhat different from dsDNA. However, it should be mentioned that the color is also influenced by pH and chemicals that interact with the stain/nucleic acid complex. The total emission wavelength depends on the filters that you use.
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