hey guys the reason for EDTA i found was to inactivate the DNase activity but,since we also add proteinase K which can take care of DNase then why we need EDTA. and if EDTA is added in intermediate step,DNase would have initially chopped off DNA.
it's not exactly my field, I don't work with DNA, but most of the cleaving proteins (as the DNase) need calcium ions to work, by "removing" (technically it just becomes unavailable) the calcium with EDTA (chelating agent for Ca and Fe ions), you inactivate quickly those proteins, which are generally quick acting.
I might be wrong, as I'm not an expert on this, but this is how I would see it...
i agree but my question is why are we adding at the intermediate step and not the initial step....if we want to stop DNase we should add EDTA at the initial step.
Hi. In CTAB method we use it in the first step (it is in the CTAB solution) but for other methods maybe we want to remove ions to gain a high quality DNA without ion (remove DNase activity, non ion DNA for PCR and ...)
A few educated guesses:
1. Might be that you need divalent ions around in the beginning to keep the gDNA in a relatively 'native' state, to assist in the prep steps. I can imagine that taking divalent ions away too early might make it much harder to have the DNA stay easily separable from the other cellular content.
2. Also, thinking that chelation of divalents might cause more protein denaturation/precipitation in the first step, and aggregates will not break down as easily with proteases.
3. If you expose cells to too much chelation, they turn into dense little raisins. I've observed that effect make cells extremely resistant to lysis --> if you turn the cells into little snots, you won't get anything out of them easily.
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