Sanger Tree of Life HMW DNA Extraction: Automated Low-Input Plant Organic HMW DNA Extraction (LoPOE) of Bryophyta v1

This protocol is a modified version of the Sanger Tree of Life’s ‘standard-input’ Automated Plant Organic HMW DNA Extraction (POE) procedure, designed specifically for Bryophyta species with low (<30 mg) or exceptionally low (<10 mg) whole-specimen tissue masses. Low-input POE (LoPOE) is divided into five stages: cell/tissue lysis with an SDS-based buffer with additives to prevent DNA degradation, centrifugation (input mass dependent) and DS-protein complex precipitation with potassium acetate, DNA isolation by one chloroform phase separation, DNA capture/purification with a 1X SpeedBead SPRI (0.45X SPRI omitted), and DNA concentration using the Eppendorf Concentrator plus. This protocol generally yields a sufficient quantity of high purity, ultra HMW (uHMW; 100 kb+) DNA for either LI or ULI library preparation, therefore DNA may be directed towards any of the Sanger Tree of Life HMW DNA Fragmentation protocols. However, outcomes are dependent on a large number of factors, including: the tissue mass input, species, tissue type, sample quality and consistency of the homogenate. Acronyms HMW: high molecular weight uHMW: ultra high molecular weight gDNA: genomic DNA SPRI: solid-phase reversible immobilisation LI: low input ULI: ultra-low input