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Lab Basics: How The Alkaline Lysis Method Works

Posted in: DNA / RNA Manipulation and Analysis
Scientist holding up alkaline pH test strip to represent alkaline lysis in the lab

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Alkaline lysis was first described by Birnboim and Doly in 1979 and has, with a few modifications, been the preferred method for plasmid DNA extraction from bacteria ever since.[1] The easiest way to describe how alkaline lysis works is to go through the procedure and explain each step, so here goes.

A Step-by-Step Guide to Alkaline Lysis

Step 1: Cell Growth and Harvesting

You start with the growth of the bacterial cell culture harboring your plasmid. When sufficient growth has been achieved, the cells are pelleted by centrifugation to remove them from the growth medium.

Step 2: Resuspension

The pellet is then resuspended in a solution (normally called solution 1, or similar in the kits) containing Tris, EDTA, glucose, and RNase A.

Divalent cations (Mg2+, Ca2+) are essential for DNase activity and the integrity of the bacterial cell wall.

EDTA chelates divalent cations in the solution preventing DNases from damaging the plasmid and also helps by destabilizing the cell wall.

Glucose maintains the osmotic pressure so the cells don’t burst and RNase A is included to degrade cellular RNA when the cells are lysed.

Step 3: Alkaline Lysis

The lysis buffer (aka solution 2) contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS).

SDS solubilizes the cell membrane.

NaOH helps to break down the cell wall, but more importantly, it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and your plasmid, to single-stranded DNA (ssDNA).

This process is called denaturation and is a central part of the procedure, which is why it is called alkaline lysis.

SDS also denatures most of the proteins in the cells, which helps with the separation of the proteins from the plasmid later in the process.

It is important during this step to make sure that the re-suspension and lysis buffers are well mixed, although not too vigorously (see below). Check out my related article on 5 tips on vector preparation for gene cloning for more information and tips. Also, remember that SDS and NaOH are pretty nasty so it’s advisable to wear gloves and eye protection when performing alkaline lysis.

Step 4: Neutralization

The addition of potassium acetate (solution 3) decreases the alkalinity of the mixture. Under these conditions the hydrogen bonding between the bases of the single-stranded DNA can be re-established, so the ssDNA can re-nature to dsDNA. This is the selective part.

While it is easy for the small circular plasmid DNA to re-nature, it is impossible to properly anneal those huge gDNA stretches. This is why it’s important to be gentle during the lysis step because vigorous mixing or vortexing will shear the gDNA producing shorter stretches that can re-anneal and contaminate your plasmid prep.

While the double-stranded plasmid can dissolve easily in solution, the single-stranded genomic DNA, the SDS, and the denatured cellular proteins stick together through hydrophobic interactions to form a white precipitate. The precipitate can easily be separated from the plasmid DNA solution by centrifugation.

Step 5: Cleaning and Concentration

Now your plasmid DNA has been separated from the majority of the cell debris but is in a solution containing lots of salt, EDTA, RNase, and residual cellular proteins and debris, so it’s not much use for downstream applications. The next step is to clean up the solution and concentrate the plasmid DNA.

There are several ways to do this, including phenol/chloroform extraction followed by ethanol precipitation and affinity chromatography-based methods using a support that preferentially binds to the plasmid DNA under certain conditions of salt or pH, but releases it under other conditions. The most common methods are detailed in the article on 5 ways to clean up a DNA sample.

So, how often do you use alkaline lysis for your plasmid preps? Let us know, in the comments section, any cool tips and tricks that you use to get better and faster results!

References

  1. Birnboim H.C. and Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research, 1979;7(6):1513–23.

Originally published October 8, 2014. Reviewed and republished June 2021.

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73 Comments

  1. Rishabh jain on December 14, 2009 at 4:01 pm

    hello nick,
    I had a Practical exam today,My teacher asked me that “what would happen if we increase the incubation period after adding 3M potassium acetate in plasmid dna isolation by alkaline lysis method”?

    As we know that potassium acetate will help in renaturation of plasmid dna,but chromosomal dna will not renature.

    will the chromosomal dna will also renature after long incubation???



  2. Rishabh jain on December 14, 2009 at 3:54 pm

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



  3. Nick on November 16, 2009 at 4:27 am

    Jode, thanks for that fantastic explanation.



  4. Jode on November 14, 2009 at 8:47 pm

    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 (https://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/) for more clarification.



  5. Ranga on November 11, 2009 at 9:00 pm

    I have a question: how does potassium acetate help here in plasmid extraction? In DNA precipitation, we add sodium acetate, the function of which would be to (1) neutralize the phosphate group making DNA less soluble (2) disrupt the hydration layer/hydrogen bonding (not sure which) by CH3COO-, again decreasing the solubility of DNA. Considering this fact, how does potassium acetate increase the solubility of plasmid? And on a separate note, forgive me if I’m dumb: how does CH3COOK *decrease* the alkalinity of the solution?



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