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We all use our favorite techniques for DNA cloning, such as Gibson assembly, TOPO cloning, ligation independent cloning (LIC), and TA cloning. However, DNA purification methods themselves, haven’t changed all that much since the 90’s. Historically, the introduction of phenol extraction in 1956, to purify nucleic acids from rat liver, rapidly replaced previous techniques.1 RNA purification methods continued to be progressively modified until 1987, when guanidinium thiocyanate-phenol-chloroform became the standard for the next 20 years.2,3 About three years later, the discovery that nucleic acids adhered to silica particles in the presence of chaotropic compounds, ushered in the era of the now ubiquitous DNA/RNA miniprep columns.4 Since then, unless you use a more obscure technique such as in-gel ligation, you’re likely doing DNA purification with a narrow set of tools.
How (else) could you get enzymes, detergents, etc. out of a solution of nucleic acid?
BIA (Benzyl:Isoamyl Alcohol), is phenol’s more docile sister, and is an organic extractant to remove PCR inhibitors from blood samples.5 You can use it to isolate DNA by combining it with the RNAse inhibitor isoamyl alcohol in a volumetric 9:1 ratio. Then use the combo just like phenol:chloroform to extract your DNA. A formal protocol would look something like this (by “something like”, I mean this is what I use exactly):
1. Add 150 ul TE to a 50 ul DNA solution to be purified (usually a digestion).
2. Add 20 ul of 9:1 (v/v) benzyl alcohol/isoamyl alcohol. Then vortex until emulsified.
3. Spin down at max speed for 3 minutes. After removal from the centrifuge, the organic layer may become cloudy. This seems to have no effect.
4. Remove the supernatant and ethanol precipitate the sample.
Isoamyl alcohol, when it’s not blocking RNAse activity and preventing foaming in phenol:chloroform mixtures, is (apparently) an extremely powerful protein precipitant. In this case, you mix it with a denser solvent which lets it pellet to the bottom of the tube when centrifuged. I’ve tested this technique with solutions of concentrated BSA and found no detectable protein left in the aqueous layer.
As a bonus, BIA seems to completely extract detergents such as Triton-X100. Also, BIA isn’t substantially denser than water since benzyl alcohol is about 1.04 mg/cm3. Adding isoamyl alcohol probably changes that some, but protein pellets (mostly) at the bottom of the tube and skips the aqueous-organic interface. Small amounts of solvent left in the aqueous layer are easily removed in subsequent ethanol washes. Also the tiny bit remaining doesn’t seem to inhibit DNA ligase.
I have yet to have my cloning fail, provided my DNA concentrations are high enough, though your mileage may vary. Two college sophomores were able to clone four constructs with a 100% success rate using the protocol. I use it for simple digestion/ligation/transformation methods, and haven’t tried any other techniques.
This protocol has only been tested for use in preparing DNA for vector cloning. Cautioned is advised, if you use it for your RNA preps, and your RT-PCR takes a trip through the woods, as your PhD money will not be refunded. BIA is first and foremost a protein precipitant and a detergent extractant. Note, the protein pellet may not be completely denatured, and accidentally re-suspending it in the aqueous phase could return zombified EcoRI into your sample. This same goes for tissue extractions, as BIA does not seem to be a strong chaotropic agent. Thus proteins may not be completely denatured.
If your solution is high in salt, BIA’s hydrophobic droplet might not pellet. This is because the density of BIA and water is similar. BIA may also not precipitate contaminants from highly chaotropic solutions such as guanidinium thiocyanate.
Note that this protocol isn’t super optimized, so publish any modifications for great DNA and RNA purifications.
1. Kirby, K.S. (1956). A new method for the isolation of ribonucleic acids from mammalian tissues. Biochem. J. 64, 405–408.
2. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.
3. Chomczynski, P., and Sacchi, N. (2006). The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nat. Protoc. 1, 581–585.
4. Boom, R., Sol, C.J., Salimans, M.M., Jansen, C.L., Wertheim-van Dillen, P.M., and van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495–503.
5. Fredricks, D.N., and Relman, D.A. (1998). Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR Inhibitor Sodium Polyanetholesulfonate. J. Clin. Microbiol. 36, 2810–2816.