May 23, 2023

Public workspaceCloning individual AAV capsid variants

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.n2bvj87ebgk5/v1
  • Sripriya Ravindra Kumar1,
  • Timothy F. Shay1,
  • Xinhong Chen1,
  • David Brown1,
  • Tatyana Dobreva1,
  • Qin Huang1,
  • Xiaozhe Ding1,
  • Yicheng Luo1,
  • Pétur H. Einarsson1,
  • Alon Greenbaum1,
  • Min J. Jang1,
  • Benjamin E. Deverman1,
  • Viviana Gradinaru1
  • 1California Institute of Technology
Miguel Chuapoco
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DOI: dx.doi.org/10.17504/protocols.io.n2bvj87ebgk5/v1
External link: https://rdcu.be/c9Vzw
Protocol Citation: 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 2023. Cloning individual AAV capsid variants. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj87ebgk5/v1
Manuscript citation:
Ravindra Kumar, S., Miles, T.F., Chen, X.et al.Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.Nat Methods17, 541–550 (2020). https://doi.org/10.1038/s41592-020-0799-7
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 08, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 81615
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
This protocol describes the procedure to clone individual AAV capsid variants for single variant characterization.
Protocol materials
ReagentpUCmini-iCAP-PHP.B plasmidaddgeneCatalog #103002
Step 1
ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 500 rxnsNew England BiolabsCatalog #M0494L
Step 3
ReagentNEB Stable Competent E.coli (High Efficiency) - 20x0.05 mlNew England BiolabsCatalog #C3040H
Step 5
ReagentMscI - 1,250 unitsNew England BiolabsCatalog #R0534L
Step 1
ReagentAgeI - 1,500 unitsNew England BiolabsCatalog #R0552L
Step 1
Digest ReagentpUCmini-iCAP-PHP.B plasmidaddgeneCatalog #103002 with ReagentMscI - 1,250 unitsNew England BiolabsCatalog #R0534L and ReagentAgeI - 1,500 unitsNew England BiolabsCatalog #R0552L



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 Temperature98 °C forDuration00:00:10 , Temperature60 °C for Duration00:00:30 and Temperature72 °C for Duration00:00:10 using ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 500 rxnsNew England BiolabsCatalog #M0494L

50s
Assemble the variant-specific fragment into the digested backbone by Gibson assembly
Transform the assembled plasmid into ReagentNEB Stable Competent E.coli (High Efficiency) - 20x0.05 mlNew England BiolabsCatalog #C3040H

Select colonies on carbenicillin/ampicillin-LB-agar plates.