History[ edit ] The term plasmid was introduced in by the American molecular biologist Joshua Lederberg to refer to "any extrachromosomal hereditary determinant. Non-integrating plasmids replicate as with the top instance, whereas episomesthe lower example, can integrate into the host chromosome. In order for plasmids to replicate independently within a cell, they must possess a stretch of DNA that can act as an origin of replication. The self-replicating unit, in this case the plasmid, is called a replicon.
Isolation of plasmid DNA The various plasmid isolation techniques that are currently in use can be divided into three phases: One of the most common bacterial host strains used for the in vivo amplification of plasmids is E.
XL1-Blue cells can be readily transformed with plasmids. Depending on the amount of plasmid DNA to be isolated, bacterial cells can be grown in various volumes of shaken culture in the presence of antibiotic s suitable for the desired selection.
Following bacterial growth, the cells can be pelleted by centrifugation in a microcentrifuge. After disposing of the supernatant, plasmid isolation from the pelleted cells can proceed principally in two different Extraction of plasmid dna By using a plasmid isolation kit. In this case the isolation of the plasmid is performed using a miniaturised chromatographic column.
Resuspension of the bacterial pellet in an isotonic solution.
In this solution, the lysis of the cells does not yet take place. Some earlier protocols also applied lysozyme to break down the bacterial cell wall.
In addition to the above listed components, the resuspension solution can also contain RNase enzyme in order to break down ribonucleic acids that are later released into the lysate.
Alkaline lysis of the cells by applying an alkaline solution of sodium dodecyl sulfate SDS that disintegrates the lipid structure of the cell membrane.
In addition, this treatment will denature both proteins and DNA, and keep these molecules dissolved in their denatured form. Precipitation of dissolved proteins, membrane debris and associated genomic DNA by applying a solution of acidic potassium acetate.
The potassium salt of dodecyl sulfate is also insoluble. Thus, this component will also precipitate. Plasmid DNA will still remain dissolved during this step. Sedimentation of the precipitated components in a microcentrifuge.
The clear supernatant will contain the plasmid DNA. Purification of plasmid DNA using a mixture of phenol and chloroform care should be taken when working with phenol as it is corrosive to human tissues. To remove the protein content of nucleic acid preparations, a mixture is often used that contains phenol and chloroform in a volume ratio of 1: Phenol denatures proteins, and chloroform readily dissolves phenol, which has limited water solubility.
When the nucleic acid preparation is shaken thoroughly with the described mixture and subsequently centrifuged, denatured proteins will be concentrated at the boundary of the upper aqueous and the lower phenol-chloroform phase of higher density.
Isoamyl alcohol reduces the frothing associated with the separation procedure. The addition of ethanol to the aqueous phase containing the plasmid will result in the precipitation of the plasmid DNA, which can thus be subsequently sedimented by centrifugation. The plasmid DNA is sedimented repeatedly by centrifugation, and then dissolved in TE solution see point e above for composition.
The preparation can be stored on ice or in a freezer. During plasmid isolation using commercially-available kits Figure The supernatant resulting from this series of steps will contain the plasmid DNA.
This supernatant is loaded on top of a mini-column containing a silicate-based membrane. The mini-column is placed in an Eppendorf tube so that the flow-through can be collected upon centrifugation.
Under the applied high ionic strength conditions, the column will bind DNA molecules in the size range of base pairs to 10 kilo-base pairs.
The column is subsequently washed with a wash buffer and a solution with high ethanol content. The ethanol is then removed via repeated centrifugation.This is a quick and efficient way to extract E.
coli plasmid DNA without using commercial kits. This technique was invented by Birnboim and Doly (). Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA.
Extraction of plasmid DNA from mammalian cultured cells Since total DNA is extracted from the cells using the kit, plasmid DNA should also be found in the extracted DNA fraction.
Therefore, we transiently transfected HEK cells with a vector carrying an Ampicillin resistance cassette (AmpR). The addition of ethanol to the aqueous phase containing the plasmid will result in the precipitation of the plasmid DNA, which can thus be subsequently sedimented by centrifugation.
The precipitate containing the plasmid DNA is washed with 70 % ethanol in order to remove the salt content of the preparation. By applying the most commonly used, so-called „miniprep” plasmid isolation protocols, µg of isolated plasmid can be prepared from mL of cell culture volume.
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