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Question about DNA conformation

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Question about DNA conformation

Postby ahkian82 » Mon Jan 22, 2007 6:40 am

Does the conformation of both native and denatured DNA show any difference? I'm looking for double-stranded DNA which is in helix form in solution. Recently, i'd bought a calf-thymus DNA from Invitrogen. In the product description, it's said that "...the DNA is prepared from highly pure, phenol-chloroform extracted DNA, and DNAse-free, RNase-free, distilled, deionized water. Once dissolved, the DNA solution is sheared to an average size of <2000bp and the concentration is adjusted to 10mg/ml..." Does the shearing process destroy the helixity of the DNA?

Upon diluting the DNA, some undissolved material was observed (the solution was not clear due to this little suspension).. Does this a sign of DNA degradation? If i remove the undissolved material via centrifugation, do you think the DNA still ok for use? (According to one of my friend, the DNA solution should be clear in the concentration that i prepared. The solution was in 4e(-4) M in Tris / DMSO (9:1) solution. Tris buffer contains 5mM Tris, 50mM NaCl in pH 7.2)

Any idea to help?? Thanks..
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Postby LilKim » Mon Jan 22, 2007 7:29 pm

Shearing does not destroy the helix... So you'd have dsDNA fragments of <2000bp.

How long did you allow the DNA to disolve? I've used ssDNA and it takes a while for it to completely dissolve (I left it overnight at 4C and shook it a couple times). Initially it was kinda 'gloopy' and cloudy. But evenutally it all disolved into solution. I also think you could use some GENTLE heat to dissolve it... (put it at 37C for 20 mins) However, i think it is unlikely that the DNA is degraded. (although i'm not sure what TRIS actually does to to DNA)

good luck!
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Postby LilKim » Mon Jan 22, 2007 8:45 pm

oh... ssDNA refers to salmon sperm DNA .. which is double stranded DNA. (Sorry for using confusing abbreviations)
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Postby ahkian82 » Thu Jan 25, 2007 9:40 am

Lilkim,

Thanks for your suggestion...haha.. It's difficult to do biological-related work in a chemistry lab.. ^_^ . We don't have the room for doing the mixing in such temperature. So, I usually prepare the solution in fresh and use it within one to two hours after preparation.. However, if i just left the solution for overnight, it's just doesn't help much in dissolving doesn't unwanted stuff..Maybe gentle heating would be a solution to this problem, I will try it out... Again, thanks so much for the suggestion.

Cheers.. :P
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Postby ahkian82 » Thu Jan 25, 2007 9:44 am

By the way, Tris is used as buffer in control the pH in physiological range, near neutral, but not below 7.2 (If i not mistaken)..
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