Protocol Online logo
Top : Forum Archives: : Molecular Biology

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

Pages: Previous 1 2 3 Next

The plot thickens... This paper from 1999 by the same people who did the regeneration of DNA columns paper describes another way of doing plasmid extraction using silica. Similar principle to the alkaline lysis process but a bit more optimised for silica binding. Anyway:

Binding of DNA to silica requires the presence of high salt concentration. We used NaCl rather than NaI (Geneclean
II, BIO 101), guanidium thiocyanate (4), or sodium perchlorate (3) to facilitate DNA binding to silica.
We tested varying concentrations of NaCl ranging from 1 to 4 M and found that 2 M and above allowed maximal
binding. Importantly, use of NaCl instead of the chaotropic salts to promote DNA binding to silica offers the
advantage that NaCl is inexpensive and the solution is stable at room temperature. Moreover, when compared
in parallel, NaCl coeluted the least quantity of bacterial RNA than NaI or guanidium thiocyanate.

Reference number 4 is the Boom paper (source of the silica-binding invention). I just read it and they didn't even try NaCl! They assumed that a chaotropic agent was required and only tried GuSCN. If only I had time to experiment with these things...


sometimes, we forget that the basis for this site is PROTOCOLS rolleyes.gif

if you go to the 'protocol' link at the top of the page, dig till you find the ones with plasmid prep solutions. that's where I got my recipes for my own P1, P2, and P3.

incidentally, back in the old days they put the recipes at the back of the manual. P3 was always KoAc/HoAc; the G Cl is a newer addition


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.


very interesting indeed.


no, trust me...P3 works fine with columns. there used to be only P3, when I started this. it was what you used when doing a maxiprep or midiprep with the Qiagen columns in the mid-90's. I suspect the alterations in the formula may improve yield? but they're not necessary. I used to get pretty fantastic yield with homemade P3, and like I said, that was when Qiagen gave you ALL the recipes in the back of the manual. at that time, they were really the only going concern for DNA prep, so they didn't have to worry so much about people stealing all their technology

gee, I'm old blink.gif


Ahh, but the midi and maxi columns are completely different to mini columns. You're right that P3 is used for midi and maxi columns (anion exchange resin), but according to qiagen you have to use proprietry buffer N3 for mini columns (silica membrane). But I believe you that buffer P3 works on mini columns, it's just that when the silica technology was invented they didn't think it would work (for some reason they never tried - have a look at the Boom paper from 1990). So the 'official' stance from Qiagen is that you have to buy buffer N3 from them if you want to use mini silica spin columns - they provide the recipes for all the other buffers *except* for N3. They also conveniently sell bottles of N3...

So based on all this it seems the 'official stance' is wrong, and that buffer P3 will also work on mini columns, or at least, buffer P3 + 2M NaCl will work. We could write a book called 'Molecular Biology Secrets "They" Don't Want You to Know About'


Could you tell me which protocol you used to regenerate the column? Is this boiling for 10 min?

QUOTE (Zouden @ May 4 2007, 05:55 AM)
Well, I've been using the protocol mentioned in this forum to reuse miniprep spin columns. The whole process is very easy and I get excellent results (no different to fresh columns) saving lots of money! But I'm running out of the solutions so I've started making them. I can't do all of them so I'd like to hear your ideas.

Qiagen are kind enough to describe the composition of some of their buffers, which are presumably the same for all brands as it's a well-established protocol.
Buffer P1 - resuspension buffer
50mM Tris-Cl, pH 8.0, 10mM EDTA, 100ug/mL RNase A

Buffer P2 - Lysis Buffer
200mM NaOH, 1% SDS

Buffer N3 - Neutralization
Q: What is the composition of Buffer N3?
A: The composition of buffer N3 is confidential. It is a proprietary component of the QIAprep Spin Miniprep Kit.

And that's as far as I got. I know that N3 contains a chaotropic salt, probably guanidine hydrochloride, to enable the DNA to bind to the silica, and probably acetic acid to bring the pH down. But what concentrations?

Any ideas? I've spent quite a bit of time looking around the net and couldn't find it.


Buffer N3 contains 25 - 50% Guanidinium Chloride and 10 - 25% Acetic Acid according to the MSDS. Note that MSDS contents do not necessarily list all components. Density is 1.14 g/cm3 and the pH is 4.3 @ 20°C.
25 –50% guanidinium chloride equals 2.6 – 5.2 M, but let’s round that off to 2.5 – 5 M. .
Glacial acetic acid is 17.4 M, so 10 - 25% would equal 1.7 – 4.4 M.
Maniatis utilizes 6 M guanidinium chloride (MW 95.53) and 0.2 M acetic acid (MW 60.05). However, 6 M guanidinium chloride = 57.32%.


I just want to write to confirm that buffer P3 - standard miniprep (KoAc pH 5.5) neutralization solution does NOT work in spin columns. The DNA will not bind.

Next I'll try P3 + 2M NaCl.


Store the used columns in 1N HCl. Wash with H2O before next use. I regenerate maxi prep column by this method, too.

[quote name='thc' date='Jul 16 2007, 12:06 PM' post='104788']
Could you tell me which protocol you used to regenerate the column? Is this boiling for 10 min?


Pages: Previous 1 2 3 Next