Plasmid Preparation DNA is specifically prepared from small amounts of the diverse cultures of the plasmid-containing bacteria.
As a result, a plasmid preparation is considered to be an important method of DNA extraction as well as purification for plasmid DNA.
In this regard, several methods have been developed for the purification of plasmid DNA from bacteria that you should know about. Generally, three steps are involved in the methods of plasmid preparation such as:
- Step 1: Growth of the bacterial culture
The first step of the plasmid preparation generally involves the growth of the bacterial culture. Plasmids are mainly purified from liquid bacterial cultures such as E.coli which have been both isolated as well as transformed.
All the plasmid vectors virtually encode 1 or more that antibiotic resistance genes as a selectable marker. Bacteria possessing a gene encoding kanamycin or ampicillin resistance helps them to multiply uninhibited.
On the other hand, the bacteria which has not taken up the plasmid vector are known to lack the resistance gene. So, only those colonies which have represented successful transformations are expected to grow, and that too under favorable conditions.
- Step 2: Lysis and harvesting of the bacteria
Whenever the bacteria are lysed under alkaline conditions, then the denaturation of both chromosomal DNA, as well as protein, occurs. But, in this case, the plasmid DNA remains stable.
To successfully reduce the ssDNA occurrence, some scientists contribute to reducing the NaOH concentration to 0.1M.
Again, the precipitation of the less supercoiled and large chromosomal DNA occurs with the addition of acetate-containing neutralization buffer. On the other hand, the small bacterial DNA plasmids remain in the solution only.
- Step 3: Plasmid DNA Purification
There are a variety of methods that are used for the purification of nucleic acid. Again, all of these particular methods work on the principle of generating specific conditions where there is either only the precipitation of the biomolecules or the nucleic acid.
As a result, this specific method helps in the separation of nucleic acid.
Importance Of Plasmid Preparation
The pure preparation of the starting materials for further experiments can be very annoying. So, in this regard, a completely new approach is to outsource your plasmid preparation. But, this should effectively suit your experimental needs.
Talking about the plasmid preparation services, these are mainly required in pharma and biotech companies along with research labs for a variety of purposes such as:
- Specific studies related to gene and cell therapy
- Production of antibodies
- Production of DNA vaccine
- Production of viral vectors
- Production of recombinant proteins
- Some other preclinical studies
So, there are many important advantages of this plasmid DNA preparation which you need to know about before opting for related services.
Therefore, the above-discussed ones are considered to be the most important things which you should know about plasmid DNA sequencing.