DNA in 80mM NaOH - (Apr/20/2005 )
Certain protocol suggesting us to add 8mM NaOH to disolve DNA. What will happen if someone add 80mM instate of 8mM NaOH? Will it degrade DNA or inhibit PCR?
I am asking this because when I first time do PCR for about 2000 bp, I managed to get it. When I did it second time, I got a 2000 bp band along with other band about 600 bp. When I did for third time, the 2000 bp band just vanished and only the 600 bp band can seen.
Is there any way for me to get rid of the 80mM of NaOH from my extracted DNA?
In 80mM of NaOH your DNA is suffering degradation. I don't remember the degradation reaction but I know DNA is not stable above certain pH. In fact PCR is showing you a slow rate DNA degradation which reflects that degradation is not mediated by an enzimatic activity (e.g. nuclease). I don't know the point to get rid of the excees of NaOH because the DNA is already degradated, but you could add the same equivalents of HCl and you'll get right pH but with some amount of NaCl which you could dialize.
i have observed plant genomic DNA that i extracted by CTAB protocol undergo this type of degradation several times, actually i had no idea what was happening? what i see on the gell is very odd. I see a nice genomic DNA band on the top, but somewhere below DNA appears like a intence blob. its not RNA i have checked. typically when DNA degrades it should appear in a countineous smear isn't it? any idea