Jul 12, 2018

Public workspaceAdenoBuilder: modular assembly of recombinant adenovirus genomes from high-copy plasmids V.1

DOI
dx.doi.org/10.17504/protocols.io.qt5dwq6
  • Jessica J. Miciak1,
  • Jason Hirshberg and Fred Bunz1,
  • Fred Bunz1
  • 1Johns Hopkins University
Fred Bunz
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DOI: dx.doi.org/10.17504/protocols.io.qt5dwq6
Protocol Citation: Jessica J. Miciak, Jason Hirshberg and Fred Bunz, Fred Bunz 2018. AdenoBuilder: modular assembly of recombinant adenovirus genomes from high-copy plasmids. protocols.io https://dx.doi.org/10.17504/protocols.io.qt5dwq6
Manuscript citation:

Miciak JJ, Hirshberg J, Bunz F.

Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

PLoS One. 2018 Jun 27;13(6):e0199563
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: June 08, 2018
Last Modified: July 12, 2018
Protocol Integer ID: 12893
Keywords: recombinant adenovirus, in vitro assembly
Abstract
The diverse applications of mutant and recombinant adenoviruses demand facile methods for their generation. This protocol allows the user to rapidly assemble customized adenoviral genomes from high-copy plasmids in a two-step isothermal reaction. Following in vitro assembly, full-length genomes can be directly packaged in HEK293 cells. Crude preparations of infectious virus can be prepared in less than one week.
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Materials
MATERIALS
ReagentPhusion Hot Start Flex 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0536S
ReagentBstBI - 2,500 unitsNew England BiolabsCatalog #R0519S
ReagentDMEM, High GlucoseLife TechnologiesCatalog #11965-092
ReagentNEBuilder HiFi DNA Assembly Master Mix - 10 rxnsNew England BiolabsCatalog #E2621S
ReagentGel Loading Dye, Purple (6X), no SDS - 4.0 mlNew England BiolabsCatalog #B7025S
ReagentLonza Nucleofector 4dLonzaCatalog #AAF-1002X
ReagentTE buffer
ReagentBuffered Phenol Chloroform Isoamyl alcohol (P:C:I) ((25:24:1, saturated with 10 mM Tris, pH 8.0 and 1 mM EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #Sigma P2069
ReagentTrypsin EDTAGibco - Thermo Fisher ScientificCatalog #25-051-CI.
ReagentBSAMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7906
ReagentParaformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS)
ReagentCostar® 24-Well Flat-Bottom Plate, Tissue Culture-Treated 50 Plates STEMCELL Technologies Inc.Catalog #38017
ReagentT25 or T75 Flask
ReagentSF Cell Line 4D-Nucleofector X Kit L (12 RCT)LonzaCatalog #V4XC-2012
ReagentFITC-conjugated anti-Hexon antibodyThermo Fisher ScientificCatalog #MA1-7330
ReagentAmmonium acetate solution for molecular biology, 7.5 MMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2706
ReagentHEK293ATCCCatalog #CRL-1573
ReagentGibco™ Penicillin-Streptomycin (10,000 U/mL)Fisher ScientificCatalog #15-140-122
Safety warnings
Adenovirus cause generally mild respiratory tract infections which are self-limiting and generally asymptomatic despite virologic and serologic proof of infection. BSL-2 facilities, equipment, and operational practices are required for work involving infectious or potentially infectious materials. Contact your institutional biosafety office for additional information.