Quantcast
Skip to content

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. Roosevelt Anyanwu on September 28, 2018 at 7:38 pm

    I could be wrong but Glucose here seems optional since the cells need to be subsequently lysed. However, in the Lysozyme method, we definitely need to prevent cell bursting.

    • Harsh on June 30, 2019 at 8:20 pm

      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

  2. suman on May 3, 2017 at 2:04 pm

    Hello
    can Someone suggest me about addition og TE- Rnase in plasmid isolation process. I have added water by mistake. What can i do now for getting DNA.

    • Sagar on January 10, 2019 at 9:35 am

      Your plasmid is still dissolves in Water so dont worry, your plasmid is there in water

  3. benish on January 31, 2017 at 9:18 pm

    plz tell me which method for DNA extraction from bacterial cells is mostly used n successful

    • aashish siddharth on June 16, 2017 at 7:37 pm

      we have a methods

  4. Vijay Kumar on December 4, 2015 at 7:13 am

    Whether NaOH is used in genomic DNA isolation? At which stage, NaOH is used in genomic DNA isolation from bacteria? please help me out.

    • AHMET on December 11, 2015 at 3:29 am

      actually , we look to generally materials

      EDTA destroys the cell structure by connecting Mg ++ ions required for protection of the cell structure and DNA degradation enzymes that inhibit the cell.

      * SDS lysis removes the lipid molecules and disrupt the structure of cell membranes interrupting.

      * Phenol-chloroform, porém and makes it move away from the sedimented DNA.

      * Pure ethanol provides the H2O molecules in the environment compete with the DNA collapse.

      * Ethanol 70%, Na + as it allows monovalent cations and the salts to dissolve away from the environment.

      • AHMET on December 12, 2015 at 3:32 pm

        an add ,
        aim of the NaOH , connected the flexible structure of the DNA . ( EFFECT OF pH )

  5. Nasir Ali on October 21, 2015 at 6:14 pm

    Hi Nick,
    I have a question ?? Can we use Alkaline extraction method for genomic DNA ?or this method is specific for plasmid DNA .

    Regards
    Nasir

    • Diana on July 1, 2019 at 2:32 am

      Check out the HotShot method (we use it for genotyping from mouse tails or ear-punches). It’s posted under Jax labs protocols & very well known, also check Cold Spring Harbor. First published in the ‘80s, I think.
      Basically, it’s heating samples in alkaline lysis buffer (NaOH & EDTA, pH around 10), heat for 30 min to 2 hours, neutralize with Tris HCl buffer (pH around 5), then use 1-2ul for PCR.

  6. Richard Cowart on July 16, 2015 at 7:51 pm

    I have used the alkaline lysis method for plasmid purification a number of times with good success. I was wondering about RNA carry over upon EtOH precipitation of the plasmids. I have been searching around and have not found any hard discussions of evidence on this issue. I notice that in the protocol Nick recommends adding RNase A prior to addition of the NaOH/SDS. But, I do not see how the RNase A would not be denatured at this step. It occurred to me that one could add RNase A + TE buffer + lysozyme + cells and they would lyse with the RNase A then degrading RNA. After an incubation period one could then add the NaOH/SDS and proceed with the protocol. But, back to the RNA question. Does anyone know if RNAs are carried over with the EtOH precipitation?

  7. Agnese Kocere on June 2, 2015 at 11:06 pm

    Hello! I was very confused by the following; Glucose maintains the osmotic pressure so the cells don’t burst

    Why try to avoid cell bursting if you want to lyse them anyway?

    Can someone clarify this, and maybe give a better explanation to why we add glucose?

    • Shishir Pandey on February 5, 2017 at 8:11 am

      Glucose is added because it acts as a ph regulator.

    • Harsh on June 30, 2019 at 8:21 pm

      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

Leave a Comment

You must be logged in to post a comment.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Scroll To Top
Share via
Copy link