DNA precipitation - (Dec/04/2009 )
Hi,
Iam trying to isolate DNA from whole blood, recently I came across two protocols, where in one, ammonium acetate was used for protein precipitation followed by isopropanol method for DNA precipitation. In yet another, ammonium acetate was used in DNA precipitation!! When searched, it was like ammonium acetate was used in both protein and DNA precipitation, if it is so, then some DNA will get precipitated during ammonium acetate protein precptatn step?
Or is it some game of pH or molarity, same issues I read about potassium acetate also
but sodium acetate is exclusively used for DNA pptn.
I request you to kindly help me in clearing this confusion
Thanks in advance
Niveda
the idea I came through is that amm.acet is anionic, and so it pulls apart the DNA strands (by disturbing hydrogen bonds between bases). this when combined with low temperature and propanol/ethnaol (non-polar solvents) forces the DNA to precipitate.
with regard to potassium acetate in plasmid extraction, its said that it makes the cell-lysis-solution a little less alkaline (how??) so that this pH is sufficient for the small plasmid DNA to reanneal and not the big genomic DNA.
and finally amm.acet for protein extraction, its said that it decreases the dielectric constant of water, thereby disturbing the hydration layer of proteins, forcing them to precipitate (with addition of isopropanol). I'm not quite sure whats the relation between dielectric constant and hydration layer
Thank you very much, i will try to read more in this regard following your suggestion
jangajarn on Dec 29 2009, 07:20 PM said:
with regard to potassium acetate in plasmid extraction, its said that it makes the cell-lysis-solution a little less alkaline (how??) so that this pH is sufficient for the small plasmid DNA to reanneal and not the big genomic DNA.
and finally amm.acet for protein extraction, its said that it decreases the dielectric constant of water, thereby disturbing the hydration layer of proteins, forcing them to precipitate (with addition of isopropanol). I'm not quite sure whats the relation between dielectric constant and hydration layer
jangajarn on Dec 29 2009, 06:20 PM said:
with regard to potassium acetate in plasmid extraction, its said that it makes the cell-lysis-solution a little less alkaline (how??) so that this pH is sufficient for the small plasmid DNA to reanneal and not the big genomic DNA.
and finally amm.acet for protein extraction, its said that it decreases the dielectric constant of water, thereby disturbing the hydration layer of proteins, forcing them to precipitate (with addition of isopropanol). I'm not quite sure whats the relation between dielectric constant and hydration layer
potassium acetate pH5.5 usually. it neutralize the solution and renature the nucleic acid which was previously denatured by NaOH. however, genomic DNA will have trouble reannealing properly as they are huge.. eventually they precipitate out of the solution. while plasmid DNA is small and will anneal well and remain in the solution.