Document Type

Article

Publication Date

2023

DOI

10.1016/j.mex.2023.102341

Publication Title

MethodsX

Volume

11

Pages

102341 (1-6)

Abstract

Extraction of high molecular weight (HMW) DNA for long read sequencing with little to no fragmentation and high purity is difficult to acquire from cyanobacterial species. Here we describe a modified method of extraction using Promega's Wizard® HMW DNA Extraction Kit to acquire high molecular weight DNA from cyanobacterial species. The protocol used in the kit is the “3.D. Isolating HMW DNA from Gram-Positive and Gram-Negative Bacteria” protocol. During a key step in the protocol, the lingering remnants of the mucilage layer of the cyanobacterial species is removed, preventing it from sticking to the DNA pellet produced. This customized modification is done between steps 11 and 12 and called METIS (maximizing extraction, transfer isopropanol step). This step drastically reduces the remaining mucilage layer, which if kept will stick to the DNA and make the DNA unsuitable for sensitive downstream next generation sequencing, like PacBio Sequencing. This protocol has been used to assemble two genomes for cyanobacteria (Synechococcus sp. and Microcystis aeruginosa) and one from a gram-negative bacterium, Lacibacter. It also allows for HMW DNA to be rapidly extracted without the use of hazardous chemicals and without extra reagents to be purchased.

  • Maximizing extraction, transfer isopropanol step (METIS) is the key modification during the step of DNA unraveling
  • METIS reduces leftover remnants of the mucilage layer in the extraction
  • High molecular weight DNA is produced with little to no fragmentation, and both a high purity and concentration

Rights

© 2023 The Authors.

This is an open access article under the Creative Commons Attribution 4.0 International (CC BY 4.0) License.

Data Availability

Article states: Data will be made available on request.

Original Publication Citation

Hept, M. A., & Greene, L. H. (2023). Extracting high-molecular weight DNA from cyanobacteria using Promega's Wizard® HMW DNA Extraction Kit with a modified protocol, METIS. MethodsX, 11, 1-6, Article 102341. https://doi.org/10.1016/j.mex.2023.102341

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