Viral vector and mouse genomic DNAs were extracted from tissues by a DNeasy Blood and Tissue Kit (Qiagen) according to the kit protocol and quantified by quantitative polymerase chain reaction (qPCR) with a TaqMan assay. The primers and TaqMan probe used for the WPRE sequence were 5′-GCAT...
TitrationqPCR, Titer meet requirementsqPCR, Titer meet requirementsdd PCR, Titer meet requirements AAV purity analysis–SDS-PAGE, Coomassie Blue Staining, Capsid band size meet referenceSDS-PAGE, Silver Staining, Capsid band size meet reference, Capsid band size meet reference ...
for 30 min at 25 °C and the reaction stopped by heating at 65 °C for 20 min. We performed qPCR on the ligated product (template) using M13 universal primers (M13f: GTAAAACGACGGCCAG, M13r: CAGGAAACAGCTATGAC), according to the following reaction mix: FastStart Essential DNA Probes Mas...
AAV qPCR titration Overview Simple workflow:optimized protocol that eliminates the need for freeze-thaw cycles and ultracentrifugation steps High purity for reliable data:purified AAV is suitable forin vivoresearch Serotype agnostic:precipitation and filtration protocol that canpurify AAV particles from any...
D'Costa S, Blouin V, Broucque F, Penaud-Budloo M, Francois A, Perez IC, et al. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR. Mol Ther Methods Clin Dev. 2016;5:16019....
AAV Vector Titration DNase1-resistant viral genomes of iodixanol-purified vector preparations were quantified by TaqMan qPCR (Thermo Fisher, catalog no. 4304449) using primer/probe set detecting CMV promoter. Vector purity was assessed by SDS-PAGE electrophoresis. AAV Transduction All luciferase transduct...
We measure the physical titer of AAV by directly extracting viral genome from lysed viral particles, and then using qPCR to accurately quantify the copy number of viral genome (using the copy number of ITR region as a proxy) in the stock. Titration services using other methods (e.g. ddPCR...
Fast, easy and reliable AAV2 titer determination with PROGEN's AAV2 Xpress ELISA: assay time was reduced to less than 2 hours by adjustment of the kit components and the protocol of our standard AAV2 Titration ELISA 2.0R seamless and risk-free transitio
The AAV vectors purified with either iodixanol or CsCl gradients were titered using quantitative real-time PCR (qPCR). For this purpose, the DNA was extracted from the AAV vectors using an alkaline lysis protocol, in which 10 µl of the AAV vector sample were mixed with 10 µl TE...
Virus was titered using RT-qPCR targeted to the ITRs, as previously described51, using a PvuII (NEB)-linearized plasmid standard. Primers used for titration are listed in Supplementary Data 2. Protein isolation and Western blot analysis Dissected muscles were snap-frozen in liquid nitrogen and ...