Do you know how to extract plasmid DNA ? - The Veterinary Site

Do you know how to extract plasmid DNA ?

Plasmid DNA Extraction
Plasmid DNA extraction is a bit trickier because plasmid DNA must be kept separate from gDNA. This separation is based on size, and good separation relies on using the right lysis method.
1. Alkaline Lysis
For plasmid DNA extraction, the lysis has to be a lot more subtle than simply chewing up the cell wall with enzymes or bashing it with glass beads. Birnboim and Doly invented the (virtually) universal method for via alkaline lysis in 1979.
The lysis buffer contains sodium hydroxide and SDS, which completely denature plasmid and gDNA (i.e. separating the DNA into single strands). It is critical that this step is performed quickly because excessive denaturing may result in irreversibly denatured plasmid.
Next the sample is neutralized in a potassium acetate solution to renature the plasmid, and this is key to the separation of plasmid and gDNA.
Because plasmids are small, they can easily reanneal forming dsDNA. Genomic DNA, however, is too long to reanneal fully, and instead it tends to tangle so that complimentary strands remain separated.
During centrifugation, gDNA (bound to protein) forms a pellet while plasmid DNA remains soluble. It is key at this step not to vortex or mix the sample vigorously because gDNA breaks easily, and broken gDNA may be small enough to reanneal and go into solution with the plasmid.
2. Purification
Plasmid DNA in the supernatant can then be ethanol precipitated or cleaned up using phenol-chloroform or a spin filter. If you are using spin filter technology, the neutralization buffer will contain guanidine salts so the lysate can bind the silica directly for further washing and elution. The resulting DNA is pure enough for most downstream molecular biology applications. If you need the plasmid for transfection, anion-exchange purification is a better choice to remove contaminating endotoxin. Endotoxin removal is also possible using faster silica-based purification setups.
This method is compatible with mammalian and other eukaryotic plasmids as well as other small extra-chromosomal DNA species. Bear in mind however that is often much lower in mammalian cells and plant extranuclear organelles.

Do you know how to extract plasmid DNA ? Do you know how to extract plasmid DNA ? Reviewed by Maaz ahmed siddiqui on 10 May Rating: 5

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