Table 1.

Troubleshooting Guide for Low-Yield AAVs

Possible Source	Troubleshoot
Packaging plasmids and the transfer vector	Check plasmid integrity with restriction enzyme digestion and/or sequencing prior to use.
Length of insert	Insert (sequences between ITRS) should not exceed 5.2 Kb. Transfer vectors with >4.5 kb package poorly. Increase starting material.
Pelleted virus	Remove supernatant from ultracentrifuge tubes carefully to maintain an intact pellet.
Insert	Check if insert is toxic to cells by monitoring transfected packaging cells.
Serotype	AAVs with serotype 2 often yields low titers. Increase starting material or increase the centrifugation time at step 29 to reduce the volume of virus.