pgegen at pgegen at
Wed Oct 25 18:00:53 EST 1995

In <4620iv$cnj at>, bbraun at writes:
>In <461ran$f85 at>, nian at writes:
>>Is anybody using acid-phenol to remove the nick or linearized DNA from circular 
>>dsDNA.  What is the critical step?  How lower the pH can be? What is the 
>>mechanism ?  I tried many times but no success at all. Any suggestions will be 
>>Nian G.

Linear DNA partitions into the phenol phase at abount pH 4.5. (That's why all the 
original RNA isolation procedures were done at pH 5.0.)

The protocol of Micard et al. (Anal. Biochem. 148:121-126 (1985)) for plasmid DNA purification includes an acid-phenol 
extraction to remove chromosomal DNA. We've used this successfully in the past; romoval of linear plasmid is not 100%
 but is pretty good. (Most of the time, however, we don't bother with the acid step.) For any phenol extraction to work 
well, you should use fresh crystalline phenol that was stored under N2;
dissolve in water and bring to 1% in 8-hydroxyquinoline to prevent oxidation. The actual extraction is best done with 
standard PCI (phenol:CHCl3:Isoamyl alchohol, 25:24:1). 

| Peter Gegenheimer                          |  pgegen at    |
| Departments of Biochemistry and of Botany  |  voice: 913-864-3939          |
| University of Kansas                       | FAX  : 913-864-5321           |
| 2045 Haworth Hall                          | "You can't fool reality."     |  
| Lawrence  KS  66045-2106                   |                   R. Feinman  |  

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