I'm trying to detect ssDNAs with acrylamide electrophoresis, which seems not working well.
I loaded 10 uL of single strand oligo DNA whose length and concentration is 20 nt and 100 uM, which equals to 6.2 µg (100 pg/µL x 10 µL = 1 nmol ≈ 6.2 µg) of amount, respectively.
I used SAFELOOK(TM) Load-Green (6×) (Wako 199-18153) as a loading dye and stain.
Unexpectedly, I did not see any bands.
I think that the amount of 6.2 µg is quite large and cannot be missed if it is double-strand DNA.
I detected a band of one of my samples, which is 47-nt ssDNA. The brightness of this band was pretty close to that of the DNA ladder, whose DNA amount was 30 ng.
I had never done detection of ssDNA by electrophoresis before, so I don't know what the problem is.
Are ssDNAs much less detectable compared to dsDNAs?
Thank you in advance.
Many dyes intercalate between the 2 strands of nucleic acid but single stranded nucleic acids only have short regions of double stranding making the dyes less effective. Silver staining is very effective but ruins the samples. With large amounts of nucleic acid shadowing can work without staining the sample. Put a fluorescent screen like a thin laye chromatograpy plate behind the gel and expose to UV light. The dna/rna absorbs some UV so you get fluorescence everywhere on the plate but dark bands where your nucleic acid id in the gel I think that x-ray intensifying screens work for this as well
Paul Rutland Thank you so much for your answer. So, ssDNA is much less stainable with intercalating stains, and I need much more amount to detect the band in acrylamide gel electrophoresis. I guess I need to think about how to do this again.
you may want to have a look at sybr gold for greater sensitivity
www.thermofisher.com/uk/en/home/references/molecular-probes-the-handbook/nucleic-acid-detection-and-genomics-technology
Paul Rutland Thank you for your suggestion! SYBR Gold could be the solution for my issue.
Daisuke Koga also be aware of 2 aspects of ssdna running. SSdna forms secondary structure in non denaturing gels so appears as the wrong size/sizes.
Almost all dna dyes have an effect on the apparent size of the dna if pre stained so if accurate size is necessary for your sample d not pre stain but run the gel first and then stain for dna
Paul Rutland Thank you for your kind answers!
I used non-denaturing gels. I guess the the RNA molecules may have formed multiple secondary structures, which made the band blurry and, for some samples, undetectable.
If I use denaturing gel, I cannot stain ssDNA with dyes that intercalate, can I?
I indeed need to distinguish multiple ssDNAs by their sizes. In this case, electrophoresis using denaturing gels and post-stain with SYBR gold would be the best way to achieve that, am I right?
Thank you in advance.
As you have probably spotted SYB Gold can stain DNA and RNA in denaturing urea, formaldehyde, or glyoxal gels So it should not be necessary even to wash the denaturant out of the gel so ,yes, I would proceed as you suggest Daisuke Koga
Paul Rutland Thank you so much for all your suggestions! I will try using denaturing gels and SYBR Gold.
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