Lab Basics: How The Alkaline Lysis Method Works
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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
- 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.
Thanks Nick God bless you
Monisha & sameerbau,
The pKa of glucose is around 12, so I suspect that the glucose was originally added to Buffer 1 to control the pH after the addition of Buffer 2, but I don’t know that for sure. Obviously Qiagen has shown us that the glucose in Buffer 1 isn’t strictly necessary.
Pooja,
This should work fine on a hypothetical RNA plasmid. In fact, RNase is often added to Buffer 1 to prevent RNA from co-purifying with the plasmid.
Lista S,
I assume the ghost band you’re describing runs just a little faster than the bulk of the DNA (negatively supercoiled) and is resistant to restriction digestion. The occurrence of this form is a function of both the time the plasmid is in the denaturing conditions as well as the temperature of the solution during that time. Rather than lowering the pH of Buffer 2, I would incubate the resuspended pellets on ice for a minute or two before adding Buffer 2 to lower the temperature of the solution during this step a little bit. After the addition of Buffer 2 and mixing, you should incubate the solution at room temperature – if you do this incubation on ice, cell lysis may not occur efficiently.
Thanks for such an informative article! I was wondering, I keep getting a “ghost” band of denatured plasmid DNA, which I know is an artifact of alkaline lysis, but I can’t seem to find the best method to completely rid my minipreps of it. The band lessens when I leave the cells in the lysis solution for only one minute, but it won’t completely dissappear. I am considering pH-ing my lysis solution down to 12 before doing my next miniprep, any ideas on whether this will cure it?
if suppose there is a dsRNA, then can this method would be applicable for extraction of this dsRNA plasmid??
Wonderful article!!! The Alkaline lysis method is crystal clear to me now, thanks to this article….
Nick could you please write an article on how Genomic DNA isolation by the CTAB method works…. I somehow never seem to get results with that method , so understanding how it works might help me to figure out why i never am able to isolate genomic dna.