Cloning individual AAV capsid variants
Sripriya Ravindra Kumar, Timothy F. Shay, Xinhong Chen, David Brown, Tatyana Dobreva, Qin Huang, Xiaozhe Ding, Yicheng Luo, Pétur H. Einarsson, Alon Greenbaum, Min J. Jang, Benjamin E. Deverman, Viviana Gradinaru
Abstract
This protocol describes the procedure to clone individual AAV capsid variants for single variant characterization.
Steps
Digest
Design 100 bp primers for the desired plasmid variant that cover the insertion region with ~20 bp overlap of the backbone
To synthesize the double-stranded DNA fragment, anneal the primers by PCR with 20 cycles of 98°C for0h 0m 10s, 60°C for 0h 0m 30s and 72°C for 0h 0m 10s using
Assemble the variant-specific fragment into the digested backbone by Gibson assembly
Transform the assembled plasmid into
Select colonies on carbenicillin/ampicillin-LB-agar plates.
