Study on the Plasmid and Genomic DNA Stability of DH5α Host System

Plasmids are extremely valuable as a source of DNA and for use in biotechnology. Our research goal was to develop plasmids with extremely high copy numbers for lab-scale plasmid preparations. Using the beverage method simplifies the process of obtaining a large quantity of plasmid DNA for various applications. Diet Coke as a beverage demonstrated that DNA recovery is highly adequate. Diet Sting with EcoRI and BamHI enzymes was used to detect restriction digestion with high sensitivity. However, for lab-scale preparative work, our findings show that plasmid yield can be significantly increased by using standard growth procedures and commonly used growth media.


INTRODUCTION
For more than a decade, geneticists have been interested in inducing mutations with chemicals and radiations with the goal of discovering mutagenic compounds through their specific activity, which can likely provide some understanding and knowledge of the chemical basis of mutation and gene structure [1]. Plasmids are extremely valuable as a source of DNA vaccines and in biotechnology applications. Increasing plasmid yield would benefit molecular biology research by lowering reagent costs and increasing experimental throughput [2]. Plasmids are used as vectors in many expression systems to produce recombinant proteins or nonproteinous recombinant components [3]. Plasmids have a significant impact on productivity, it is necessary to conduct research on plasmid stability and colony forming units. at both the industrial and research levels [4]. Plasmids can also perform additional functions, such as plasmid-encoded genes that can improve the host's survival by completely killing a different organism or synthesizing toxins that act as defensive mechanisms. [5]. A single organism may contain multiple plasmids, each with a distinct function [6]. Restriction digestion is a technique that involves cutting a specific region of genetic material and incubating the targeted sequence with a restriction enzyme that binds to specific DNA sequences and cleaves at specific nucleotides [7]. Restriction digestion allows for the ligation of genetic material fragments, which is required for biological research [8].

Plasmid Preparation
Single-cell colonies were picked and streaked out on selective LB-agar plates and further transferred to 2 mL selective LB-medium for 24 h at 37°C. The master LB-agar plate was stored at 4°C, whereas the overnight culture was used for plasmid Mini

Restriction Analysis
Analytical restriction digestions with specific restriction endonucleases (Fermentas or New England BioLabs) were performed for verifying the expected plasmid by means of the DNA fragments analyzed with gel electrophoresis. After visualizing the correct band pattern for the estimated plasmid size within one or more clones, the respective clone was used for inoculation of approximately 100 mL selective LB-medium and incubated overnight at 37°C under shaking [10].

Restriction Analysis
In this gel plasmid and genomic DNA is seen affected by the different types of beverages. By doing this we observed that only Diet Coke and Sting were affected so Sting and Diet Coke was undergone restriction digestion for specific identity with EcoRI, BamHI and HindIII [ Fig. 1A].
In this gel, genomic DNA is seen affected by the different types of beverages. By doing this we observed that only the Diet Sting and Sting were affected so Sting and Diet Sting was undergone restriction digestion for specific identity [ Fig. 1B].
We have observed only digestion in the Diet Sting EcoRI, BamHI and HindIII enzymes [ Fig. 1C].

Quantification of Gel Band
The public domain Image J tool was used to analyse the ECL image (developed at the National Institutes of Health and available at http://rsb.info. nih.gov/ij/), using the "measure" functions. The results of the analysis are a value for each band which is proportional to the Integrated Density Value (IDV) of the band. All images were processed in such a way that no information was lost.  Sting (EcoRI) ------------8 Sting (HindII1) ------------9 Sting (BamH1) --------

CONCLUSION
With the choice of several beverages in the isolation of genomic and plasmid DNA of bacterial cells, the choice of Diet Coke as a beverage proved that the recovery of DNA is highly adequate and however the role of these beverages to be further studied to understand the recovery process and any changes or modifications to be known if various other recombinant technology methods are carried out.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.