What a Good Library Looks Like Justin O’Grady
DNA preparation Fresh cells and fresh extraction best gTip, manual, automated – include RNase Phenol chloroform clean up of DNA Ethanol precipitation High molecular weight good - >60,000 kb Ensure well resuspended/homogenous Anything stuck in the well?
Shearing 3ug to start 5000-6000rpm on an Eppendorf 6-8kb average 0.4x Ampure bead wash Tapestation – check size Look for small fragments Repeat 0.4x wash if necessary
Post-shearing
Post 0.4x wash
FFPE Worthwhile? 1x Ampure wash Tapestation
Pre sequencing mix 8-15 ng/ul Tapestation Size should remain similar Not too much small stuff
General tips Wash steps throughout are critical Main source of loss of material Magnetic rack important Avoid bead loss/carryover Rely on Qubit for quantification and Tapestation for quality Nanodrop useful for quality but not quantification
GOOD LUCK!!