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

Mohamed A Elnagar; Mohamed F Ibrahim; Magdy Albert; Maya M Talal; Mahmoud M Abdelfattah; Ehab El-Dabaa; Reham Helwa

doi:10.1186/s13568-022-01483-x

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

AMB Express. 2022 Nov 1;12(1):137. doi: 10.1186/s13568-022-01483-x.

Authors

Mohamed A Elnagar^#¹, Mohamed F Ibrahim^#¹, Magdy Albert¹, Maya M Talal¹, Mahmoud M Abdelfattah², Ehab El-Dabaa³, Reham Helwa⁴

Affiliations

¹ Biotechnology program, Faculty of Science, Ain Shams University, Cairo, Egypt.
² Molecular Cancer Biology Group, Zoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt.
³ Center of Excellence in Recombinant Pharmaceutical Proteins, Biochemistry and Molecular Biology Department, Theodore Bilharze Research Institute, Cairo, Egypt.
⁴ Molecular Cancer Biology Group, Zoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt. Reham.helwa@sci.asu.edu.eg.

^# Contributed equally.

Abstract

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.

Keywords: DNA ladder; Miniprep; Plasmid; RNase A.