Hello,
I don't understand the difference in what you can see after running your DNA on an urea or an alkaline gel. I know how alkaline works: high pH, hydrogen bonds are prevented, as a result your DNA migrates as ssDNA. But as far as I understand, it is the same with urea: you denature secondary structures with urea, but in the end it is also used to purify or analyze ssDNA.
I already run multiple urea PAGEs and I could distinguish between different ssDNA molecules on the gel. Now my supervisor told me to run an alkaline gel. However, I do not understand what additional information it would give me, since both are denaturing gels? How do you decide to run an urea or an alkaline gel?
Formamide and urea are very versatile but can make the gel rubbery. Strong alkali may cause hydrolysis of the gel so the speed of separation changes between the first and second half of the gel while the urea runs more evenly. THis may be an advantage depending on the sizes that you want to separate It may be that the medium is changed rather than the nucleic acid
Sometimes some DNA species show differential denaturation in urea vs alkaline gels. So, I am guessing your supervisor wants to cross check the denaturation patterns.
I looked into the paper from 1988 describing the method. Article A Simple Technique for Quantitation of Low-Levels of DNA Dam...
"Thus, it is clear that alkaline conditions will permit a more sensitive detection of DNA damage, including single- and double-stranded DNA breaks and alkali-labile regions, such as apurinic and apyrimidinic sites [9] and phosphotriesters [10]"Well, not identifying only breaks but also...
I know that people in the neighboring DNA repair group were dealing with the repair of abasic sites.
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