User:Nabaa099/Plasmid preparation

= Plasmid Preparation = A plasmid preparation is a method of DNA extraction and purification for plasmid DNA, it is an important step in many molecular biology experiments and is essential for the successful use of plasmids in research and biotechnology. 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

Harvesting and lysis of the bacteria
There are several methods for preparing plasmids, including alkaline lysis, mechanical lysis, and enzymatic lysis.

Alkaline lysis
The most common method is alkaline lysis, which involves the use of a high concentration of a basic solution, such as sodium hydroxide, to lyse the bacterial cells. When bacteria are lysed under alkaline conditions (pH 12.0–12.5) both chromosomal DNA and protein are denatured; the plasmid DNA however, remains stable. 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 to lower the pH to around 7, the large and less supercoiled chromosomal DNA and proteins form large complexes and precipitate; but the small bacterial DNA plasmids stay in solution.

Mechanical lysis
Mechanical lysis involves the use of physical force, such as grinding or sonication, to break down bacterial cells and release the plasmid DNA. There are several different mechanical lysis methods that can be used, including French press, bead-beating, and ultrasonication.

Enzymatic lysis
Enzymatic lysis, also called Lysozyme lysis, involves the use of enzymes to break down the bacterial cell walls and release the plasmid DNA. The most commonly used enzyme for this purpose is lysozyme, which breaks down the peptidoglycan in the cell wall of Gram-positive bacteria. Lysozyme is usually added to the bacterial culture, followed by heating and/or shaking the culture to release the plasmid DNA.

Preparations by size
Plasmid preparation can be divided into five main categories based on the scale of the preparation: minipreparation, midipreparation, maxipreparation, megapreparation, and gegapreparation. The choice of which method to use will depend on the amount of plasmid DNA required, as well as the specific application for which it will be used.

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, they are: 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 plasmid DNA from bacteria. Commonly used miniprep methods include alkaline lysis and spin-column based kits. 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 5 to 50 µg depending on the cell strain. Miniprep of a large number of plasmids can also be done conveniently on filter paper by lysing the cell and eluting the plasmid on to filter paper.

Midipreparation
A midiprep, also called medium-scale preparation, is used to prepare larger amounts of plasmid DNA, usually in the range of a few tens of milligrams to a few grams. Midiprep methods are typically more efficient than miniprep methods, and they are suitable for applications such as cloning, protein expression, and library construction. Some commonly used midiprep methods include alkaline lysis, spin-column based kits, and gradient purification. The starting E. coli culture volume is 15-25 mL of Lysogeny broth (LB) and the expected DNA yield is 100-350 µg.

Maxipreparation
A maxiprep, also known as large-scale preparation, is used to prepare very large amounts of plasmid DNA, usually in the range of tens of grams to several kilograms. Maxiprep methods are typically the most efficient and are used in applications such as large-scale protein production, genome sequencing, and production of plasmid vectors. Some of the most common maxiprep methods include alkaline lysis and silica-based spin-column kits, and Qiagen maxiprep kit. The starting E. coli culture volume is 100-200 mL of LB and the expected DNA yield is 500-850 µg.

Megapreparation
A megaprep, known as mega-scale preparation, is a method for preparing even larger amounts of plasmid DNA than a maxiprep, typically in the range of hundreds of grams to several kilograms. It is similar to maxiprep, but with a larger scale, therefore it will use similar techniques as maxiprep and some variations can be adjusted to optimize the yield. They are typically used for large-scale applications such as production of viral vectors for gene therapy, production of plasmid DNA for vaccination and large-scale library construction. The starting E. coli culture volume is 500 mL – 2.5 L of LB and the expected DNA yield is 1.5-2.5 mg.

Gigapreparation
A gegaprep is even larger than a megaprep, used to purify DNA in the range of tens of kilograms or even more. Gegapreps are used for huge scale applications such as genome sequencing, large-scale protein production, and the production of plasmid DNA for therapeutics and vaccines in an industrial setting. They are typically carried out in a well-equipped laboratory with large-scale equipment and can take weeks to complete. Gegapreps methods are mainly based on silica-based spin-column kits and column chromatography, the steps are similar to maxiprep but with a much larger scale. The starting E. coli culture volume is 2.5-5 L of LB and the expected DNA yield is 7.5–10 mg.

Purification of plasmid DNA
It is important to consider the downstream applications of the plasmid DNA when choosing a purification method. For example, if the plasmid is to be used for transfection or electroporation, a purification method that results in high purity and low endotoxin levels is desirable. Similarly, if the plasmid is to be used for sequencing or PCR, a purification method that results in high yield and minimal contaminants is desirable. However, multiple methods of nucleic acid purification exist. All work on the principle of generating conditions where either only the nucleic acid precipitates, or only other biomolecules precipitate, allowing the nucleic acid to be separated.

Ethanol precipitation
Ethanol precipitation is a widely used method for purifying and concentrating nucleic acids, including plasmid DNA. The basic principle of this method is that nucleic acids are insoluble in ethanol or isopropanol but soluble in water. Therefore, It works by using ethanol as an antisolvent of DNA, causing it to precipitate out of solution and then it can be collected by centrifugation. The soluble fraction is discarded to remove other biomolecules.

Spin column
Spin column-based nucleic acid purification is a method of purifying DNA, RNA or plasmid from a sample using a spin column filter. The method is based on the principle of selectively binding nucleic acids to a solid matrix in the spin column, while other contaminants, such as proteins and salts, are washed away. The conditions are then changed to elute the purified nucleic acid off the column using a suitable elution buffer.

Phenol–chloroform extraction
The basic principle of the phenol-chloroform extraction is that DNA and RNA are relatively insoluble in phenol and chloroform, while other cellular components are relatively soluble in these solvents. The ddition of a phenol/chloroform mixture will dissolve protein and lipid contaminants, leaving the nucleic acids in the aqueous phase. It also denatures proteins, like DNase, which is especially important if the plasmids are to be used for enzyme digestion. Otherwise, smearing may occur in enzyme restricted form of plasmid DNA.

Beads-based extraction
In beads-based extraction, addition of a mixture containing magnetic beads commonly made of iron ions binds to plasmid DNA, separating them from unwanted compounds by a magnetic rod or stand. The plasmid-bound beads are then released by removal of the magnetic field and extracted in an elution solution for down-stream experiments such as transformation or restriction digestion. This form of miniprep can also be automated, which increases the conveniency while reducing mechanical error. Moreover, some beads-based plasmid mini prep reagents are 100% centrifuge-free and use bacteria culture medium directly.

Solid-Phase extraction with Carbon Nanotubes
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