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The Basics: How Alkaline Lysis Works

plasmid-prep.jpgAlkaline lysis was first described by Birnboim and Doly in 1979 (Nucleic Acids Res. 7, 1513-1523) and has, with a few modifications, been the preferred method for plasmid DNA extraction from bacteria ever since. The easiest way to describe how alkaline lysis works is to go through the procedure and explain each step, so here goes.

1. Cell Growth and Harvesting

The procedure starts 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.

2. Re-suspension

The pellet is then re-suspended in a solution (normally called solution I, 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.

3. Lysis

The lysis buffer (aka solution 2) contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS). SDS is there to solubilize 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 central part of the procedure, which is why it’s 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 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.

4. Neutralization

Addition of potassium acetate (solution 3) returns 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 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.

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 chromotography-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 my article on 5 ways to clean up a DNA sample.

73 Comments

  1. EduHerNav on May 10, 2013 at 9:25 pm

    Great! thanks a lot for sharing.

  2. samkjm on February 19, 2013 at 2:41 pm

    Why use alkali and not acid to lyse cells???

    • Diana on July 1, 2019 at 2:28 am

      DNA is more stable in higher pH, RNA prefers slightly acidic. The high pH won’t hurt you DNA, but it helps separate out proteins & RNA, in a way.

  3. earl on September 20, 2012 at 11:10 am

    Awesome article! Thank you! 🙂

  4. JANI_MAULIK on May 27, 2012 at 8:01 am

    i don’t think glucose in slo. 1 is to prevent cell burst,even in water these cells won’t burst..

    i might be wrong..

    • Harsh on June 30, 2019 at 8:22 pm

      You are right Maulik.
      Since ultimate purpose is to break the cell, there is no logic to use glucose to maintain isotonocity. However, glucose plays important role. Purpose of adding glucose is to maintain the pH of the solution between 12 to 12.5 . Glucose has pKa of 12.3. At this pH, genomic DNA denatures and plasmid DNA remains intact.
      Ref. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC342324/?page=2

  5. laurie on October 24, 2011 at 3:27 pm

    Hello!
    i’m working on a alkaline extraction for gram+ gram- and yeast, for set up after a reat time for diagnose urinary tract infection. i would like to do a purifiation on column after all for an eventual automatisation. Curently i try to do this with Qiagen( qiagen DNA mini kit)silica column and their buffer and their protocole.i explain myself, after i extract my DNA i put AL ( chaotropics salts) i heat the all for 10 minutes at 70 degrees and after that put the all on the silica column and wash it with many buffer ( AW1,AW2…). in my extraction protocole they are 70% of ethanol. when i make my real time and i compare my method with qiagen method( proteinase K ATL= detersif ) i have a Cp of 14 with Qiagen method and 18 with mine for a bacteria culture of 10^8 at first. without the Qiagen column i have a Cp of 14 with my procedure. if someone have an idear?

    thank you a lot guys

  6. bumunang on September 19, 2011 at 3:26 pm

    Thanks Nick God bless you

  7. Jode Plank on August 4, 2011 at 4:24 am

    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.

  8. Lista S on August 1, 2011 at 5:49 pm

    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?

  9. pooja on July 20, 2011 at 2:27 pm

    if suppose there is a dsRNA, then can this method would be applicable for extraction of this dsRNA plasmid??

  10. Shreya Roy on May 9, 2011 at 11:50 am

    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.

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