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

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.

The old Qiagen handbook should have the exact composition. I dont have it. Anyone has the old handbooks?

I doubt it would. That was quote from the Qiagen website, describing buffer N3 as 'confidential' and 'proprietary'.

Given that the Qiagen kit and pretty much every other miniprep kit is based on the old alkaline lysis method, I reckon there's a strong chance that N3 would be mostly potassium acetate...

How about an some guesswork

It is a known fact that the solution used in a spin column are basically alkaline lysis solutions. The N3 solution corresponds to Solution III (alkaline lysis) with addition of guanidine hydrochloride to make the DNA bind to the column.

The formulation for solution III:
3M KOAc
2M HOAc

And the amount of guanidine hydrochloride used in DNA extraction is:
4M Guanidine hydrochloride

Thus guessworks says
3M KOAc
2M HOAc
4M Guanidine hydrochloride

Give a try and report back if this works.

Is it possible to reuse gel extraction columns also? If so then is the same method applied as is for miniprep columns?

Yes. both silicon based DNA binding columns use the same principle and more or less the same material.

Recent manuals suggest a wash with 35% guanidinium HCl, which is 350 g/l. 4M GuHCl is only 282 g/l. You might want to try 4.5 - 5 Molar.

Ahh, so we've narrowed it down to a few different concentrations!
Now I'm wondering, the original chaotropic salt principle used guanidinium isothiocyanate which is more expensive, and I think I read somewhere on this forum that it's a more effective chaotropic agent than GuHCl so presumably less of it is needed.

If I have time to try it I will! But if anyone else can try it, I'm very interested to hear your results...