Good afternoon! We are trying to do bisulfite conversion in a new way and we want to evaluate DNA integrity after bisulfite conversion by agarose gel electrophoresis. For tests, we use 5 µg of mouse liver DNA. At the output, we get a concentration of 50 ng/µl. For electrophoresis, we take 4 µl of converted DNA bisulfite. We use 1% agarose gel, 1x TAE buffer and ethidium bromide okara. The duration of electrophoresis is 1 hour at a voltage of 120. After electrophoresis, a five-minute incubation on an ice bath enabled partial hybridization and visualization of the DNA. But we don't see any bands. Perhaps someone can tell what we are doing wrong?
Why do we expect the post process dna to form bands rather than a smear. You are running 200ng of dna and if this is randomly broken up then 200ng may be spread over 10 cm of gel. Did you see the untreated dna control sample on the same gel?. I would expect that ,on average, 20ng spread over 1cm of gel would be almost invisible. Was an equivalent amount of starting dna visible in the gel?
Are you satisfied that there is no rna in your starting material that would cause an overestimate in the amount of dna
Paul Rutland We don't even see smear, mouse liver genomic DNA (which we use for bisulfite conversion) at the same concentration is perfectly visualized. Maybe we should put more bisulfite converted DNA on the gel?
Bearing in mind that much of your product will be single stranded ( in spite of your cold step) so traps less ETBR and also the reannealing is poor due to the base changes induced I think that it will take a lot of dna to give a visible signal. You could try running more dna and do not run it very far at first so it is more concentrated in the gel which may give a better idea of the level of degradation caused by the procedure. You can always put the gel back in the tank and run it further if you can see the product on the short run Nataliia Karpova
Thank you very much for your advice, I will try and do it Paul Rutland
Here are some potential reasons why you may not be seeing any bands after electrophoresis:
I hope these suggestions help you identify the issue and improve your results.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
- Badges
- Science method