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Can any one clarify my question with DNA

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Can any one clarify my question with DNA

Postby shivakumar » Fri Mar 14, 2008 6:29 am

hi everybody

while extracting DNA, extraction buffer containing
NaCl, EDTA, Tris n CTAB
whts d role of those chemicals n
n why adjusting d buffer pH to 8

n I feel same doubt with chloroform isoamyl alcohol(their role)
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Postby canalon » Fri Mar 14, 2008 3:25 pm

I can clarify your question. I would use good syntax and complete words. It would like that:

"In a protocol for the extraction of DNA we use the following reagents, could you tell me what are their different roles? NaCl, EDTA, Tris, CTAB, Chloroform/Isoamyl alcohol.
Similarly why is the pH adjusted to 8?"

Now as a beginning of an answer:
NaCl is used to prevent the co-precipitation of DNA and CTAB.
EDTA is used to chelate divalent cations, which are necessary (among other things) for proper DNASe activity
Tris is a Buffer
CTAB is a cationic detergent that precicpitates polysaccharides
Chloroform is usually used with phenol to denature proteins. IAA is just here to improve the separation of the aqueous and polar phases
DNA degrades in acidic environment (depurination)

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
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Postby Cat » Fri Mar 14, 2008 4:12 pm

pH 8 as far as I know is optimal for the extraction and was determined experimentally (correct me if I am wrong).

NaCl and CTAB - used together to remove polysaccharides
EDTA - chelating agent, used to remove Mg2+ and Ca2+ from DNase enzymes and prevent DNA degradation.
TRIS - keeps DNA soluble in water by keeping it deprotonated
Phenol:chloroform isoamyl alcohol - to remove proteins
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