What is special about the N3 buffer for Qiagen minipreps?

Question

For those who use Qiagen miniprep columns, they may be aware that the P3 buffer is the acetic acid step to neutralize cell lysis. They may also be aware that the Qiagen P3 buffer cannot be interchanged with the proprietary N3 buffer. It is also known that the N3 buffer is particularly unique since it primes the miniprep columns for binding. I was wondering about that priming step and what chemically primes the columns. Would it be appropriate to use QBT buffer to prime the miniprep columns?

Answer 1

The Qiagen manual for the Miniprep Kit states that buffer N3 contains guanidine hydrochloride and acetic acid. The MSDS for buffer N3 gives a bit more information:

• Guanidinium hydrochloride 25-50%
• Acetic acid 10-25%
• pH 4.3

Buffer P3 uses 3M potassium acetate, the potassium is usually important in plasmid preparations as potassium SDS is used to precipitate the proteins in solution.

This information might be enough to create a buffer that should work with the silica-gel columns.

I also found a patent about a method for isolating nucleic acids that states that silica particles can bind nucleic acids in the presence of chaotropic salts, so the guianidinium hydrochloride is probably the important difference in the N3 buffer.

The patent for this method from Qiagen goes into even more detail, according to this the buffer composition is:

• N3: 4.2M guanidinium hydrochloride, 0.9M potassium acetate, pH 4.8

Answer 2

N3 and P3 are different. N3 contain chaotropic agents. P3 does not. Mainly there are two types of DNA purification kits on the market: silica membrane (glass fiber) based and DEAE anion exchange based. The silica membrane based method requires high concentration of chaotropic agents (salts that can change the water structure dramatically, GuHcl, GuSCN, NaI, are a few of them) present in the mixture before loading on to the spin column. DEAE based method, on the other hand, bind DNA based on the charge property on the DEAE resin which largely depends on the pH and ionic strength of the buffer. Chaotropic agents are not needed for DEAE based method. In our lab we make our own kit using components and buffer recipe provided by Epoch Life Science for both mini prep and maxi prep. Their mini spin column and maxi DEAE column can be purchased without buffer. Their mini spin column also works perfectly with buffer from Qiagen and Lifetech. That's how we used up our leftover buffer from our old kits.