Homemade plasmid Miniprep solutions for affordable research in low-fund laboratories

• Published in: AMB Express Vol. 12; no. 1; p. 137
• Main Authors: Elnagar, Mohamed A., Ibrahim, Mohamed F., Albert, Magdy, M.Talal, Maya, Abdelfattah, Mahmoud M., El-Dabaa, Ehab, Helwa, Reham
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.11.2022; Springer Nature B.V; SpringerOpen
• Subjects: Biomedical and Life Sciences; Biotechnology; Buffers; Cloning; College students; COVID-19; DNA ladder; E coli; Laboratories; Life Sciences; Lysis; Microbial Genetics and Genomics; Microbiology; Miniprep; Molecular biology; Original; Original Article; Pandemics; Plasmid; Plasmids; Proteins; RNase A; Plasmid; DNA ladder; RNase A; Miniprep
• ISSN: 2191-0855; 2191-0855
• DOI: 10.1186/s13568-022-01483-x

As a consequence of Covid-19 pandemic, the basic lab consumables are in shortage, especially in the low-income countries. Thus, the main objective of the present study is to develop and evaluate homemade solution to isolate plasmid. To pursue this objective, RNase A was overexpressed in Bl21 DE3 cells ( E. coli strain) and prepared as crude refolding reaction with proper activity. Also, lysis buffers, neutralization buffer, and washing buffers were prepared. The homemade miniprep kit showed successful isolation of the px48SpCas9 plasmid. The prepared plasmid purity was enough to be used successfully in PCR amplification. In addition, to get extra benefits from this study, seven primers were designed to match the plasmid backbone to produce DNA ladder (100–1500 bp). In conclusion, we were able to have attainable working solutions for plasmid miniprep and DNA ladder.