Simplified cocktail mix and PCR conditions for amplification of extracted DNA from common bacteria, fungi and algae isolates for Microbiological studies

Real-time quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) methods have revamped environmental microbiology, providing data about targeted nucleic acids of specific microorganisms found within the environment. These data are useful for the characterization of the interacting processes of targeted microbial communities. They also assess contaminant microbes within the environment (water, air, fomites). Amplification of DNA for identification of bacteria fungi or algae commonly isolated in microbiological studies is a common and constant problem in molecular analysis this may be due to various unforeseen problems like the concentration of the DNA and the Primer, the annealing temperature used or the presence of contaminate in the PCR mixture etc This study was done to address such issues. Here, we suggest suitable cocktail mixture protocols that can be successfully used to make high-quality qPCR and dPCR measurements of microorganisms in the environment yielding amplicons with good integrity, quality and concentrations that can be used for further analysis. DNA of varying concentrations from different samples were diluted using proposed ratios with water and used for PCR runs. The resulting amplicons were checked for their qualities and used for Sanger sequencing. The amplicons produced were of were of good quality and quantity which were successfully used for Sanger sequencing, giving sequences that were successfully blasted and found to be similar to sequences deposited in the NCBI repository with 90% similarity and above. The suggested protocols provide defined and direct mixing aliquots to be used in PCR mixtures for good amplification outcomes when working with DNA of varying concentrations.