Plasmid preparation
Encyclopedia
A plasmid preparation is a method used to extract and purify plasmid DNA
. Many methods have been developed to purify plasmid DNA from bacteria. These methods invariably involve three steps:
, usually E. coli
, which have been transformed
and isolated. Virtually all plasmid vectors in common use encode one or more antibiotic resistance
genes as a selectable marker
(Ex :kanamycin,Ampicillin), which allows bacteria that have been successfully transformed to multiply uninhibited.
Bacterials are grown under favourable conditions.
s are precipitated. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate
-containing neutralization buffer
the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.
DNA
from bacteria
. It is based on the alkaline lysis
method invented by the research
ers Birnboim and Doly in 1979. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep".
Minipreps are used in the process of molecular cloning
to analyze bacterial clones
. A typical plasmid DNA yield of a miniprep is 20 to 30 µg depending on the cell strain.
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
. Many methods have been developed to purify plasmid DNA from bacteria. These methods invariably involve three steps:
- Growth of the bacterial culture
- Harvesting and lysis of the bacteria
- Purification of plasmid DNA
Growth of the bacterial culture
Plasmids are almost always purified from liquid bacteria culturesMicrobiological culture
A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested,...
, usually E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
, which have been transformed
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
and isolated. Virtually all plasmid vectors in common use encode one or more antibiotic resistance
Antibiotic resistance
Antibiotic resistance is a type of drug resistance where a microorganism is able to survive exposure to an antibiotic. While a spontaneous or induced genetic mutation in bacteria may confer resistance to antimicrobial drugs, genes that confer resistance can be transferred between bacteria in a...
genes as a selectable marker
Selectable marker
A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a...
(Ex :kanamycin,Ampicillin), which allows bacteria that have been successfully transformed to multiply uninhibited.
Bacterials are grown under favourable conditions.
Harvesting and lysis of the bacteria
When bacteria are lysed under alkaline conditions both DNA and proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s are precipitated. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate
Acetate
An acetate is a derivative of acetic acid. This term includes salts and esters, as well as the anion found in solution. Most of the approximately 5 billion kilograms of acetic acid produced annually in industry are used in the production of acetates, which usually take the form of polymers. In...
-containing neutralization buffer
Buffer solution
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...
the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.
Preparations by size
Kits are available from varying manufacturers to purify plasmid DNA, which are named by size of bacterial culture and corresponding plasmid yield. In increasing order, these are the miniprep, midiprep, maxiprep, megaprep, and gigaprep. The plasmid DNA yield will vary depending on the plasmid copy number, type and size, the bacterial strain, the growth conditions, and the kit.Minipreparation
Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmidPlasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
from bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
. It is based on the alkaline lysis
Alkaline lysis
Alkaline lysis is a method used in molecular biology to break cells open to isolate plasmid DNA or other cell components such as proteins. Bacteria containing the plasmid of interest are first grown, then lysed with a strong alkaline buffer consisting of a detergent sodium dodecyl sulfate and a...
method invented by the research
Research
Research can be defined as the scientific search for knowledge, or as any systematic investigation, to establish novel facts, solve new or existing problems, prove new ideas, or develop new theories, usually using a scientific method...
ers Birnboim and Doly in 1979. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep".
Minipreps are used in the process of molecular cloning
Molecular cloning
Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
to analyze bacterial clones
Cloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...
. A typical plasmid DNA yield of a miniprep is 20 to 30 µg depending on the cell strain.