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!!