A simple approach for effective shearing and reliable concentration measurement of ultra-high-molecular-weight DNA

Pipetting and concentration measurement of viscous ultra-high-molecular-weight (UHMW) DNA samples is challenging and often highly imprecise. Effective guidelines for handling UHMW samples are missing in the field. Herein, a simple and low-cost workflow is presented that enables accurate pipetting and reliable concentration measurement. Central to the workflow is the shearing of representative small aliquots of UHMW DNA samples to a fragment size <150 kb by vortexing them for 1 min with a glass bead in a round-bottomed 2-ml tube. Additionally, a solution is provided for accurate quantitation of high-molecular-weight DNA with fluorometric (Qubit [Thermo Fisher Scientific, MA, USA]) methods by using an appropriate genomic DNA standard, resulting in values that match spectrophotometric (Nanodrop [Thermo Fisher Scientific]) optical density readings. METHOD SUMMARY This study provides guidance for handling ultra-high-molecular-weight DNA samples. A low-cost shearing approach based on vortexing with a glass bead is introduced that reduces the DNA fragment size of small representative aliquots to <150 kb, enabling reliable pipetting and concentration measurement. Additionally, the importance of using genomic DNA as standard for accurate Qubit fluorometric concentration measurement of high-molecular-weight DNA is demonstrated.