Alkaline lysis or alkaline extraction is a method used in

molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...

to isolate plasmid DNA from bacteria.

Method

Bacteria containing the

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...

of interest are first

cultured Culture () is an umbrella term which encompasses the social behavior, institutions, and norms found in human societies, as well as the knowledge, beliefs, arts, laws, customs, capabilities, and habits of the individuals in these groups.Tylor ...

, then a sample is

centrifuged A centrifuge is a device that uses centrifugal force to separate various components of a fluid. This is achieved by spinning the fluid at high speed within a container, thereby separating fluids of different densities (e.g. cream from milk) or ...

in order to concentrate cellular material (including DNA) into a pellet at the bottom of the containing vessel. The supernatant is discarded, and the pellet is then re-suspended in an

EDTA Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid with the formula H2N(CH2CO2H)2sub>2. This white, water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble complexes eve ...

-containing physiological

buffer Buffer may refer to: Science * Buffer gas, an inert or nonflammable gas * Buffer solution, a solution used to prevent changes in pH * Buffering agent, the weak acid or base in a buffer solution * Lysis buffer, in cell biology * Metal ion buffer * ...

. The purpose of the EDTA is to chelate divalent metal cations such as Mg²⁺ and Ca²⁺, which are required for the function of DNA degrading enzymes ( DNAses) and also serve to de-stabilise the DNA phosphate backbone and cell wall. Glucose in the buffer will maintain the osmotic pressure of the cell in order to prevent the cell from bursting. Tris in the buffer will retain the pH of the cell with 8.0 and RNase will remove the RNA which will disrupt the experiment. Separately, a strong

alkaline In chemistry, an alkali (; from ar, القلوي, al-qaly, lit=ashes of the saltwort) is a base (chemistry), basic, ionic compound, ionic salt (chemistry), salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as ...

solution consisting of the detergent

sodium dodecyl sulfate Sodium dodecyl sulfate (SDS) or sodium lauryl sulfate (SLS), sometimes written sodium laurilsulfate, is an organic compound with the formula . It is an anionic surfactant used in many cleaning and hygiene products. This compound is the sodium sal ...

(SDS) and a strong base such as

sodium hydroxide Sodium hydroxide, also known as lye and caustic soda, is an inorganic compound with the formula NaOH. It is a white solid ionic compound consisting of sodium cations and hydroxide anions . Sodium hydroxide is a highly caustic base and alkali ...

(NaOH) is prepared and then added. The resulting mixture is incubated for a few minutes. During this time, the detergent disrupts cell membranes and allows the alkali to contact and denature both

chromosomal A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...

and plasmid DNA. After tearing apart the cell membrane by SDS, the cell content will neutralize the NaOH; this is why the pH of the lysis goes down from 12.8 to 12.3. So if there are not enough bacterial cells, the extra NaOH will function to generate small DNA fragments. But 0.5 M L-arginine, which can supply a stable pH, can be used to replace 0.1 M sodium hydroxide. Finally,

potassium acetate Potassium acetate (CH3COOK) is the potassium salt of acetic acid. It is a hygroscopic solid at room temperature. Preparation It can be prepared by treating a potassium-containing base such as potassium hydroxide or potassium carbonate with acetic ...

is added. This acidifies the solution and allows the renaturing of plasmid DNA, but not chromosomal DNA, which is precipitated out of solution. Another function of the potassium is to cause the precipitation of

and thus removal of the detergent. A final centrifugation is carried out, and this time the pellet contains only debris and can be discarded. The plasmid-containing supernatant is carefully removed and can be further purified or used for analysis, such as

gel electrophoresis Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...

Other uses

Also, alkaline lysis is sometimes used to extract plant genetic material. The plant cells are subjected to a strongly alkaline solution containing a detergent (usually a

zwitterion In chemistry, a zwitterion ( ; ), also called an inner salt or dipolar ion, is a molecule that contains an equal number of positively- and negatively-charged functional groups. : With amino acids, for example, in solution a chemical equilibrium wil ...

ic or

nonionic An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...

detergent A detergent is a surfactant or a mixture of surfactants with cleansing properties when in dilute solutions. There are a large variety of detergents, a common family being the alkylbenzene sulfonates, which are soap-like compounds that are more ...

such as

Tween 20 Polysorbate 20 (common commercial brand names include Kolliphor PS 20, Scattics, Alkest TW 20, Tween 20, and Kotilen-20) is a polysorbate-type nonionic surfactant formed by the ethoxylation of sorbitan monolaurate. Its stability and relative non ...

), and the mixture is incubated at high temperature. This method is not used as often due to the sodium hydroxide's tendency to damage genetic material, reducing DNA fragment size.

References

Alkaline Lysis at Mama Ji's Molecular Kitchen
* Bacteriology Cell biology Molecular biology Microbiology {{cell-biology-stub