He he, in our lab this one is known as “The Snot Extraction”

Hi sir,this is amazing source for alkaline lysis method,thanks for making such a wonderful source to help students.Best up luck.

Thanks for writing these articles Nick. This kind of info. is scarce thus much appreciated. Can stop hassling the senior postdoc with these kinds of questions now!

does any concentration of NaOH react with CO2 in air…because in lab v generally prepare 10N NaOH and store it for months..is that recommended??

What does the white flocculant consist of?

why were the bacteria grown overnight in medium containing selective antibiotic?

I hope this imparts a little extra insight. Sloppy technique can incorporate relative amounts of the flocculent precipitate which may include compromised gDNA. As well as mechanical breaks residual endonucleases could degrade ss or ds gDNA into small fragments. Prolonged exposure to ethanol can dehydrate the small segments of DNA causing denaturing of double strands. Small segments of ss DNA with repeat sequences can form hairpin structures which could be incorporated into the plasmid DNA extract. (Nucleic Acids Research. 15, 8739-8754). Please feel free to correct me should I have misunderstood.

Hi Richa. The NaOH is around pH 12. This will de-protonate (remove + charge from) the Hydrogen bonds between the nucleic acid bases in the core of the double stranded DNA… allowing the strands to disassociate into single strands. Should this not occur and a “flocculant” of chromosomal DNA and proteins not clump together you will take up ds chromosomal DNA and proteins with your plasmid and it will be contaminated for whatever your doing with it next. Glacial acetic acid is just pure acetic acid. Our lab uses sodium acetate for nuetraliztion rather than potassium, (probably cheaper lol). Maybe your protocol also adds a salt or is relying on free ions in the solution. I encourage you to read the article by Birnboim and Doly that Nick Oswald cited in his main article for further clarification.

Hi Monisha. It’s not necessary providing you have a good buffer. However I can see it’s purpose in preventing cells bursting whilst you are resuspending the cell pellet. If cells burst then, you may induce some mechanical shearing of the plasmid DNA with the pipette. Another factor might be controling the timing of the de-naturing of the DNA. Knowing the first exposure the DNA receives to any de-naturing compound is when you add that NaOH/SDS. Peace of mind I guess.

Ranga,

Everything Nick described in Step 4 is true, but there is something else going on here as well. SDS is actually a salt, and when it dissolves in an aqueous solution the sodium disassociates from the dodecyl sulfate. The detergent, a long hydrophobic chain with a negatively charged sulfate group at one end, then goes on to find other hydrophobic molecules to associate with, namely the interiors of membranes and proteins, disrupting the structures of both. Solution Three contains free potassium ions, however, and potassium dodecyl sulfate is an insoluble salt (in the presence of excess K+). Once Solution Three is added to the dodecyl sulphate-denatured proteins and membranes, the potassium binds to the sulfate groups and neutralizes the charge, and the resulting KDS precipitates with the proteins and membranes in tow.

(I was surprised to read that Vlokar’s lab uses sodium acetate in Solution Three – in this situation I would expect that all precipitation of protein is occurring via a pH mediated mechanism and that the resulting clarified supernatant would still contain some dodecyl sulfate and more protein than if potassium acetate were used in Solution Three.)

If you would like to test what I mentioned above, though, simply mix a SDS solution with a KCl (or KCH3COO) solution in an eppendorff tube. You can increase the solubility of the KDS by heating it, and decrease the solubility by incubating on ice. This is why an incubation on ice and a 4°C centrifugation is common in plasmid prep protocols, particularly for protocols utilizing DEAE resins downstream, where residual dodecyl sulfate will interfere with DNA binding to the resin.

You are right that the acetate that hitched a ride with the potassium into solution three won’t neutralize the hydroxide from solution two. However, solution three has been pH’d to ~5.5 with acetic acid. Since the concentration of hydroxide is relatively low (~0.1M), the addition of a high concentration of a weak acid is able to neutralize it without damaging the DNA, as would happen if the NaOH were neutralized by a strong acid like HCl. (This would create local environments of very low pH during mixing, which would cause nicks and breaks in the DNA.)

The addition of all these salts does nothing (in a practical sense) to the solubility of the plasmid DNA because we are still in a solution with a high dielectric constant. During ethanol or isopropanol DNA precipitation, the cation (usually Na+) is only able to neutralize the negative charge on DNA and precipitate it once the dielectric constant of the solution is lowered by the addition of the alcohol. See Nick’s article on DNA precipitation (http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/) for more clarification.

hello nick,
you have cleared my year long doubts about isolation of the DNA.
thanks for doing such a wonderful job.

Jode.. Thanks for that fantastic explanation. I’m just wondering how you guys can come up with amazing explanations like this. Are you a chemist? I get so many such questions, and I do come up with answers. But what I lack is the confidence.. I’m not sure how good my reasons are. I search the internet, and if I don’t find an answer, that’s it. That question is lost in obscurity.

WHY ALKALINE LYSIS SOLUTION NEED TO PREPARE FRESHLY.

Excuse me, Why doesn’t DNA precipitated during phenol/chloroform extraction?
Both phenol/chloroform and EtOH has low Dielectric constant?

Ranga,

I’m a biochemist, which is kinda-like a chemist. Outside of that most of what I’ve learned came about after having something go horribly wrong, and then trying to figure out why. I’ve also been pretty lucky and frequently have had people around me that could help fill in the gaps in my knowledge. Hopefully, this web site can help others who don’t have that go-to person down the hall.

Yoo,

While phenol and chloroform have low dielectric constants, they are almost completely immiscible in water. Therefore, the dielectric constant of the aqueous phase (where the DNA is) is minimally effected during an extraction. Since small alcohols are miscible in water, they can drop the dielectric constant and precipitate DNA.