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Making miniprep solutions for spin columns - Now that we can reuse the columns... (May/04/2007 )

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The acetic acid is probably largely in the form of potassium acetate, since the potassium ion is required to precipitate the SDS as KDS (the white precipitate) which traps the genomic DNA and cell debris during the post-neutralization spin. Measuring the pH of the neutralized lysis product would tell you a lot about the required amount of acetic acid.


QUOTE (Zouden @ May 13 2007, 02:29 AM)
Buffer P3 is for regular miniprep, not for spin columns. For spin columns you use buffer N3 which is a 'secret' although not a very well-kept one.

Have you used buffer P3 (KoAc/HoAc) in a spin column? Did it work, or did your DNA wash through the column and not bind?

Actually, it seems like it probably would work (someone should tell Qiagen):
Biotechniques. 2000 Jan;28(1):107-9. High-throughput method for isolating plasmid DNA with reduced lipopolysaccharide content.
Isolating plasmid DNA from bacteria is a fundamental step in molecular biology. It is often accomplished by an alkaline lysis of bacteria and the subsequent adsorption of nucleic acids to silica oxide in the presence of chaotropic substances. Here we show that the addition of such chaotropic reagents is not required for the efficient DNA isolation with silica oxide. This surprising finding allowed us to purify plasmid DNA with significantly less lipopolysaccharides (LPS), which is otherwise a common bacterial contaminant of silica oxide-isolated DNA and inhibits subsequent applications. In addition, we have implemented a precipitation step that altogether leads to a reduction of the LPS content by a factor of 900 relative to published methods. Our novel protocol facilitates an inexpensive high-throughput analysis of pure plasmids in a 96-well format without the addition of hazardous reagents.
Pretty interesting, don't you think?
We stepwise reduced the concentration of guanidine hydrochloride, but even when we excluded this chemical altogether, we did not observe a significant drop in the yield of plasmid DNA (Table 1). We also found that chaotropic substances are not required for DNA binding to other silica-based DNA adsorption matrixes such as commercially available spin columns (data not shown).
The DNA binding is dependent on the presence of silica oxide and potassium acetate in the reaction (data not shown). Possibly, the high potassium acetate concentration used to neutralize the alkaline lysis of bacteria is sufficient to facilitate the DNA binding to silica.

For removal of endotoxins you can also use EndoTrap blue. It is available in different kit sizes. More information you find on


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