Plasmid DNA Isolation Procedure
DNA isolation aims to separate the plasmid DNA from the chromosomal DNA and the cellular RNA of the host bacteria, known as E.Coli. The most common procedure in isolating DNA, especially for forensic and molecular analysis is cell lysis. In cell lysis, the plasmid DNA is separated from the chromosomal DNA through phenol extraction or alkaline solution. This method is also known as cell disruption because the DNA is forced to expose from within as the fatty membrane or nucleus breaks through the process known as centrifugation.
To have an idea of how DNA isolation is done, below are the procedures involve in a simple cell lysis.
- Cell collection – Collect DNA samples from animals or plants. For animals, DNA can be found in almost all cell types, such as muscles, reproductive cells, hair roots, and skin cells. An exception is the blood cells because do not have a nucleus.
- Cell growth and harvesting – Achieve a sufficient growth of bacterial cell culture by placing the cell in LB media containing ampicilin and leaving it overnight. The bacteria will grow and replicate the plasmid at the same they replicate their genome.
- DNA destruction – Pour NaOH and sodium dodecylsulfate (SDS) to the solution. SDS is a type of detergent that will help break cell wall and cell membrane, spilling out the bacteria (including proteins, fats, and carbohydrates) from the contents. The NaOH will work to denature the DNA into single strands.
- DNA precipitation – Add sodium acetate or a mixture of acetic acid and potassium acetate to neutralize the solution. Once the pH of the solution is neutralized, the DNA strands are renatured allowing the membrane material and genomic DNA to precipitate, forming like a phlegm-like mass. The plasmid DNA remains in the solution.
- DNA extraction/ purification – Separate or extract the plasmid DNA from its associated proteins by transferring the precipitate to a microcentrifuge tube (this process is also known as centrifugation).Pour isopropanol or a 50/50 mixture of phenol and chloroform to dissolve hydrophobic molecules and denature proteins. The nucleic acid will precipitate; therefore purifying DNA from protein contaminants.
Other procedures in isolating DNA include alcohol precipitation and protease or RNase (as for RNA removal) addition. Depending on the purpose and volume of solution, the isolation process used by scientists may vary from each other. Below are several methods available to isolate plasmid DNA.
- Silica-based isolation or binding plasmid to silica with high concentrations of chaotropic salt
- Ion exchange chromatography over DEAE-modified cellulose membranes
- Polyethylene glycol precipitation
- Organic plasmid DNA extraction using phenol
- Plasmid DNA precipitation from ethanol solutions