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URL: https://dx.doi.org/DOI:10.17504/protocols.io.p5mdq46

Authors: Louise Judd

Group: High molecular weight DNA extraction from all kingdoms

Summary: This is the protocol I have developed to genterate High'ish' Molecular Weight gDNA from gram negative bacterial speices to use for ONT MinION sequencing.This protocol was intially developed for the high throughput extraction of bacterial gDNA for Illumina short read sequencing. To perform our high throughput work we utislise Beckman Coulter Biomek liquid handling robots and the Beckman Coutler gDNA extraction kit Genfind v2. This kit utilises magnetic pariticles and is relatively gentle. We have found that gDNA extracted using this method has a high'ish' molecular weight. On a fragment analyser the GNDA is typically a clean single peak >60 kb (the limits of the fragment analyser). ONT MinION libraries generated wiht this gDNA typically have N50s in excess of 20 kb and the data is suitable for the assembly automated of almost bacterial genomes we have tested to date. This protocol will not give you the ultra long 'whale' reads that others have reported but it is a good compromise between HMW DNA, speed and ease of use and capacity to scale up to high throughput. NOTEWe have found that gDNA extracted using this method often have underepresentation of smaller plamids (

Affiliations: University of Melbourne

Version: 1

Publication Date: 2018

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