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From: whycare@athena.mit.edu (nemo)
Newsgroups: sci.bio
Subject: <None>
Message-ID: <1990Jul3.034956.25953@athena.mit.edu>
Date: 3 Jul 90 04:52:39 GMT
Sender: news@athena.mit.edu (News system)
Reply-To: whycare@athena.mit.edu (nemo)
Organization: Massachusetts Institute of Technology
Lines: 47

Subject:More Questions on DNA Purification 
Date: Tue, 3 Jul 90 03:49:56 GMT
Lines: 43

1. How would the salt in a mixture of DNA, protein, and 70% ethanol affect the
   charge on the protein? I ask this because I'm still wondering why the phenol
   denatures the protein (i.e. how the phenol disrupts the noncovalent 
   interactions between peptide chains)

2. Does the phenol disrupt disulfide bridges? 

3. What is the approximate pH of a mixture of DNA, protein, 70% ethanol, and
   2.5 M ammonium acetate?

4. Does the ethanol partition into the organic phase in a standard protein 
   extraction?

5. Why doesn't the DNA partition into the organic phase? Someone explained that
   it was due to the phosphodiester bonds of the DNA, but that doesn't explain 
   how the charges on the molecule interact with the charges on the
phenol and       the chloroform.  

6. Specifically, why is protein amphipathic? What parts of proteins,
what            structural motifs allow it to behave in such a manner?

7. What is the charge distribution on phenol and chloroform molecules?
I'm           getting confused assuming that there is positive charge
delocalizing into the
   phenol ring and that the carbon molecule of chloroform is extremely
positive.
   These assumptions bother me, because it seems that if they were true, some
   DNA would get trapped in interface along with the protein.  This
leads me to      the next questions:

8. What would happen if no salt were used in the purification of DNA? Would a
   lot of DNA get trapped right below the organic phase along with the
protein?    
9. Is high salt concentration solely used to precipitate the DNA? Or is it used
   to stabilize the molecule as well?  Indeed, if the salt were used only to
   precipitate the DNA, then it hardly seems necessary to put DNA in a salt 
   solution before the protein were removed if DNA were inherently hydrophobic
   to the organic phase. The addition of the salt would only be necessary when
   one wanted to force the DNA into a concentrated pellet.

Any answers to the above questions are welcome.
Mechanistic details are very happily received.
Send e-mail to whycare@athena.mit.edu.