1. What a Good Library Looks Like
Justin O’Grady

2. 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?

4. 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

5. Post-shearing

6. Post 0.4x wash

7. FFPE
Worthwhile?
1x Ampure wash
Tapestation

9. Pre sequencing mix 8-15 ng/ul Tapestation Size should remain similar
Not too much small stuff

11. 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

12. GOOD LUCK!!