Published: June 16th, 2017 Last Modified: November 23rd, 2019
Silica based nucleic acid purification columns and kits are a common consumable in the lab. They’re used for cleaning up PCR reactions, purifying gel slices and plasmid DNA minipreps. Inevitably, when you reach the bottom of the box you are usually left with one of two scenarios: 1) You don’t have enough of a specific buffer or 2) You don’t have enough columns. Both of these scenarios usually result in the remaining components of the kit being tossed out or forgotten in your cupboard for a decade (that somehow make their way into my hands).
Why not source your own columns, mix your own buffers to both reduce waste and save money! Grab a snack, ease into your bunny slippers, this is a long one.
There are two major technologies when it comes to purifying DNA column-style; silica based purification and anion-exchange resin purification. In this post I’ll be talking about silica kits, but the anion exchange post is coming too.
Silica Based Kits
Silica purification is by far the most common technology due to the low cost of materials, and is generally found in kits for smaller scale purifications, such as miniprep kits, PCR cleanup kits and gel purification kits. Silica will bind DNA in the presence of a high concentration of chaotropic salts such as guanadinium hydrochloride (Gu-HCl) or sodium iodide (NaI). The silica is then washed a few times with a high concentration of ethanol and finally eluted in a low volume of pH 8-8.5 Tris.
So, what if you run out of columns for a particular kit? Well, as long as it’s based on silica/chaotropic salt technology, you can use them interchangeably. One caveat here is that different manufacturers deposit differing amounts of silica into their columns, which translates to a higher or lower binding capacity of DNA. Also, silica columns can dry out, significantly impacting binding capacity. Dried up columns can be re-humidified and reused.
Don’t have old silica columns in a drawer somewhere? You can buy the silica spin columns by themselves much cheaper than you could get from Qiagen/GE/etc. Enzymax is the cheapest at 0.38$ per column, with Epoch Life Sciences at 0.64$ per piece. Other manufacturers do exist but I have had good experiences with both companies.
Now, what if you have the columns, but are missing a specific buffer? Don’t be scared! Since these silica kits all use the same underlying technology, the buffers themselves are interchangeable and vary little in their composition. Openwetware has the compositions of the majority of Qiagen buffers, taken straight from their manuals and patents. I’ve also scoured patents from other manufacturers for their recipes so that you can see how similar they are. The trick here is to use the appropriate buffer for your specific application.
For silica kits you will generally use three types of buffers: binding buffers that allow your DNA to bind to the silica, wash buffers which clean away unbound DNA/RNA/protein/salt, and elution buffers which dissolve your DNA that you can then use.
Qiagen Buffer P1: 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 50 ug/mL RNase A
Qiagen Buffer P2: 200 mM NaOH, 1% SDS
Binding buffer for PCR product/enzymatic reaction cleanup:
Qiagen Buffer PB :5 M Gu-HCl, 30% isopropanol, add 5 volumes to 1 volume of PCR/enzymatic reaction, load onto column.
Qiagen Buffer PB (Enzymax variant): 5 M Gu-HCl, 20 mM Tris-HCl pH 6.6, 30% ethanol
The isopropanol/ethanol is likely added here to prevent smaller DNA fragments (i.e. primers) from binding to the membrane. You could likely adjust the concentration of isopropanol to exclude binding of larger pieces. Machery Nagel does an excellent job demonstrating this in their PCR cleanup manual, although they dilute their PCR binding buffer with water.
Binding buffer for agarose gel slice purification:
Qiagen Buffer QG: (5.5 M guanidine thiocyanate, 20 mM Tris HCl pH 6.6) Add 300 uL of buffer to 100mg of gel slice, heat to solublize, load onto column. Enzymax suggests same recipe for their columns.
Promega Gel Buffer: (6M guanidine thiocyanate, 10 mM EDTA) Recipe from their patent. EDTA used to prevent discoloration of guanidine. Use ~333 uL of buffer per 100 mg of gel slice, heat to melt, load onto column.
Binding/neutralization buffers for plasmid minipreps:
Qiagen Buffer N3: 4.2 M Gu-HCl, 0.9 M potassium acetate pH 4.8. This is an interesting buffer which was gleaned from the patent, Qiagen calls it proprietary. Essentially it is Solution 3 from your classic alkaline lysis miniprep with Gu-HCl (1.73M final conc.) thrown in to get the plasmid to bind to the silica. Resuspend cells in 250 uL Buffer P1, lyse with 250 uL Buffer P2, neutralize with 350 uL of Buffer N3, load unto column.
Qiagen Buffer N3 (Enzymax Variant): 4M Gu-HCl, 0.5M Potassium Acetate pH 4.2
I wanted to put my money where my mouth is so I performed a miniprep where I used Buffer N3. After neutralization I split the lysate into equal portions and loaded them onto 4 different brands of silica spin kits I had lying around, Biobasic, Qiagen Qiaprep, Sigma Genelute and Sigma nucleic acid binding columns. After elution and reprecipitation, I ran equal volumes on a gel. As you can see this buffer will work with all of them, with differences in yield between them. As mentioned previously one factor is the differing amount of silica deposited into each tube. As well, the Sigma kits actually state that you have to run “Preperation buffer” through the columns before loading your sample. This preparation buffer is likely ~5M Gu-HCL to prepare the silica for binding. This would likely boost yeild in all kits though, just an extra step. Will add to this if I end up testing it. Also, age of kit MAY play a factor here, the Qiagen and Sigma columns are easily 10 years old vs maybe 3 years for the Biobasic.
Learn from my mistake, I had to remake this buffer because I thought I’d be smart and increase the concentration of KOAc to 1.32M as is standard in older solution 3 recipes. Everything starts crashing out with beautiful crystals. 4.2M Gu-HCl and 0.9M KOAc pH 4.8 is about the limit. It took <1 mL of HOAc to pH a 50 mL solution, easy to over do.
GE Neutralization buffer (4.4M Gu-HCl, 0.65M potassium Acetate
and 3.1M Glacial Acetic Acid). Got this from a GE healthcare patent, slightly higher Gu-HCl (2.2M final conc.) content. The acetate/acetic acid portion is very close too, you’d be adding that much acetic acid to potassium acetate to make it pH 4.8 as in the Qiagen buffer. GE uses 350 uL of this buffer for 175 uL of lysis buffer (200mM NaOH, 1%SDS) + 175 uL resuspension buffer (100 mM Tris-Cl pH7.5; 10 mM EDTA; 400ug/ml RNase A). Slightly different resuspension buffer, but identical lysis buffer.
Wash buffers for PCR/Enzymatic cleanup, gel purification/plasmid preps:
Qiagen Buffer PB: 5 M Gu-HCl, 30% isopropanol. This is an optional wash buffer used to remove residual nucleases/carbohydrates that may be stuck to the silica, recommended when isolating plasmids from endA+ strains. Wash once with 500 uL. Proceed with regular washing steps.
Qiagen Buffer PE: (10 mM Tris-HCl pH 7.5, 80% ethanol) Wash twice with 750 uL.
5X Qiagen Buffer PE (Enzymax Variant): (80 mM NaCl, 8mM Tris-HCl pH 7.5) Dilute to 1X with 100% ethanol, 1X conc: 16 mM NaCl, 1.6 mM Tris-HCl pH 7.5, 80% EtOH.
GE Wash buffer: (2mM Tris-Cl pH8; 0.2mM EDTA and 80% ethanol) Very similar to Qiagen, would work interchangeably. GE uses 400 uL for two washes, but 750 uL like Qiagen is probably better.
Promega Wash Buffer: 80% Isopropanol
Elution buffers for PCR/Enzymatic cleanup, gel purification/plasmid preps:
Qiagen Buffer EB: (10 mM Tris·Cl, pH 8.5)
Qiagen Buffer EB (Enzymax Variant): (10 mM Tris-HCl, pH 8.0)
Machery Nagel AE Buffer: (10 mM Tris·Cl, pH 8.5)
GE Elution Buffer: (10 mM Tris-HCl, pH 8.0)
Any high pH (8-8.5) buffer will work, as will water at a lower efficiency. The presence of EDTA can also lower recovery. The amount of buffer used to elute will dictate recovery, more buffer will recover more DNA but at a lower concentration.
So, those are the buffers you need to use any brand of silica spin kit. As you can see most of the magic is in the binding buffers, which when applied to your sample have a final Gu-HCl/thiocyanate concentration of ~1.5-4M. As long as your sample has that concentration, the DNA will bind to the silica. Wash buffers are 80% ethanol/isopropanol, with Tris/EDTA added for flavour. Elution buffers are even simpler, with basically all manufacturers disclosing it to be Tris pH 8-8.5.