Rpl3 and Rpl3l cDNA were generated from liver and heart RNA, respectively, using First-Strand Synthesis SuperMix (Invitrogen, #18080400). Firefly luciferase cDNA was cloned from the pGL3 luciferase reporter plasmid (Promega). The cDNAs were then cloned into the pAV-cTnT AAV expression vector (Vigene Biosciences) utilizing the NEBuilder HiFi DNA Assembly polymerase (New England BioLabs, #E2621L). For primers, see
Nebuilder hifi dna assembly polymerase
Manufactured by New England Biolabs
NEBuilder HiFi DNA Assembly polymerase is a high-fidelity DNA polymerase designed for efficient and accurate DNA assembly. It enables seamless cloning of multiple DNA fragments in a single reaction.
Automatically generated - may contain errors
2 protocols using nebuilder hifi dna assembly polymerase
1
Adeno-Associated Viral Vector Delivery of Ribosomal Proteins
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Rpl3 and Rpl3l cDNA were generated from liver and heart RNA, respectively, using First-Strand Synthesis SuperMix (Invitrogen, #18080400). Firefly luciferase cDNA was cloned from the pGL3 luciferase reporter plasmid (Promega). The cDNAs were then cloned into the pAV-cTnT AAV expression vector (Vigene Biosciences) utilizing the NEBuilder HiFi DNA Assembly polymerase (New England BioLabs, #E2621L). For primers, seeSupplementary Tables S1 . The viruses were produced at the Howard Hughes Medical Institute Janelia Farms Viral Tools facility using the AAV9 capsid. Intrathoracic injection (50 µL volume, 28 G needle) using titers of 1012 viral genome copies was performed in 9-day-old mouse pups that were immobilized by cold anesthesia prior to injection (Wakimoto et al., 2016 (link)).
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Rpl3 and Rpl3l cDNA were generated from liver and heart RNA, respectively, using First-Strand Synthesis SuperMix (Invitrogen, #18080400). Firefly luciferase cDNA was cloned from the pGL3 luciferase reporter plasmid (Promega). The cDNAs were then cloned into the pAV-cTnT AAV expression vector (Vigene Biosciences) utilizing the NEBuilder HiFi DNA Assembly polymerase (New England BioLabs, #E2621L). For primers, see
2
Recombinant MyoAAV-PDK4 Virus Production
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Pdk4 cDNA was cloned from genomic cDNA generated from mouse heart tissue using CloneAmp HiFi polymerase (Clontech Laboratories). The cDNA was then cloned into the pAAV-MCS expression vector (Cell Biolabs Inc.) utilizing the NEBuilder HiFi DNA Assembly polymerase (New England BioLabs) (for primers, see key resources table ). MyoAAV-PDK4 virus was produced in-house using the MyoAAV 1A capsid as previously described.43 ,44 (link) Briefly, AAV Pro 293T cells (Takara) were plated in 15 cm dishes at a density of 2 ×107 cells/dish. The next day, each plate was transfected with 10.5 μg helper plasmid, 5.25 μg of the Rep/Cap plasmid, and 5.25 μg of the PDK4 MyoAAV-1A plasmid. Recombinant virus was harvested from the cells and media, and purified by ultracentrifuge using an iodixanol gradient.43 AAV titers were quantified by Taqman-based qPCR with C1000 Touch Thermal Cycler (Bio-Rad Laboratories). MyoAAV-luciferase was used as the control virus. For delivery, virus was delivered 4xE1013 (link) vg/Kg of mice via retro-orbital injection at 2-month of age.
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