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Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)

Authors :
Xi Wang
Ling Zhao
Xiaoxing Wu
Huaxiu Luo
Di Wu
Meng Zhang
Jing Zhang
Mikhail Pakvasa
William Wagstaff
Fang He
Yukun Mao
Yongtao Zhang
Changchun Niu
Meng Wu
Xia Zhao
Hao Wang
Linjuan Huang
Deyao Shi
Qing Liu
Na Ni
Kai Fu
Kelly Hynes
Jason Strelzow
Mostafa El Dafrawy
Tong-Chuan He
Hongbo Qi
Zongyue Zeng
Source :
Genes and Diseases, Vol 8, Iss 3, Pp 298-306 (2021)
Publication Year :
2021
Publisher :
KeAi Communications Co., Ltd., 2021.

Abstract

Plasmid DNA (pDNA) isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research. Almost all pDNA purification involves disruption of bacteria, removal of membrane lipids, proteins and genomic DNA, purification of pDNA from bulk lysate, and concentration of pDNA for downstream applications. While many liquid-phase and solid-phase pDNA purification methods are used, the final pDNA preparations are usually contaminated with varied degrees of host RNA, which cannot be completely digested by RNase A. To develop a simple, cost-effective, and yet effective method for RNA depletion, we investigated whether commercially available size selection magnetic beads (SSMBs), such as Mag-Bind® TotalPure NGS Kit (or Mag-Bind), can completely deplete bacterial RNA in pDNA preparations. In this proof-of-principle study, we demonstrated that, compared with RNase A digestion and two commercial plasmid affinity purification kits, the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps. Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits. We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples. Furthermore, the Mag-bind-based SSMB method costs only 5–10% of most commercial plasmid purification kits on a per sample basis. Thus, the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.

Details

Language :
English
ISSN :
23523042
Volume :
8
Issue :
3
Database :
Directory of Open Access Journals
Journal :
Genes and Diseases
Publication Type :
Academic Journal
Accession number :
edsdoj.8a99184a4fc44f8c95b21a3344f464f4
Document Type :
article
Full Text :
https://doi.org/10.1016/j.gendis.2020.04.013