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Cat. No.: UATK-50 (for 50T)

Cat. No.: UATK-200 (for 200T)

Description

Universal AAV Titration Kit by qPCR, also named Universal qPCR-Based AAV Titer Quantification Kit, enables rapid, precise and high-throughput titration of multiple Adeno-associated Virus (AAV) serotypes via quantitative real-time PCR (qPCR). Formulated with high-quality UDG enzyme and dUTP at optimized ratios, the kit effectively eliminates false-positive results and artificially low Ct values triggered by carryover contamination of previous PCR amplicons.

Adeno-associated Virus (AAV) is a small single-stranded DNA virus belonging to genus Dependovirus, family Parvoviridae, comprising over 100 distinct serotypes. After cellular transduction, AAV rarely integrates into host genomic DNA yet drives long-term stable transgene expression, prominently in a wide spectrum of non-dividing cell populations. Benefiting from high achievable titers, negligible in vivo immunogenicity upon injection, and robust infectivity toward both dividing and quiescent cells, AAV has been extensively adopted across diverse biomedical research fields.

UDG (Uracil-DNA Glycosylase), also referred to as UNG (Uracil-N-glycosylase), catalyzes hydrolytic cleavage of the N-glycosidic bond linking uracil (dU) to deoxyribose within uracil-containing DNA to release free uracil, predominantly deployed for prevention of PCR carryover contamination. Its anti-contamination mechanism is as follows: dUTP substitutes dTTP and incorporates into newly synthesized PCR amplicons during amplification to generate dU-containing DNA products. In subsequent qPCR runs, UDG selectively digests contaminating residual single- or double-stranded dU-bearing DNA carried over from prior reactions, thus eliminating interference from leftover amplicons.

Components

• DNase I (1U/μl)
• Reaction Buffer (10X)
• DNA Extraction Solution
• Enzyme Mix
• Stop Solution
• SYBR Green qPCR Mix (2X, with UDG)
• AAV Primer Mix (10μM)
• Positive Control
• Low ROX (50X)
• High ROX (50X)
• Ultrapure Water

Features

• Broad Applicability: Targeting AAV2 inverted terminal repeats (ITRs) harbored in transgene transfer plasmids for amplification and quantification, this kit is compatible with titer testing of all AAV serotypes. AAV serotype is defined by capsid protein conformation, which is dictated by the Cap gene used for viral packaging. Recombinant AAV of variant serotypes is routinely generated via triple-plasmid transfection of HEK293 cells: transfer plasmid carrying transgene plus AAV2 ITRs, helper plasmid encoding the target serotype’s Cap and AAV2 Rep genes, and adenoviral helper plasmid. Accordingly, all recombinant AAV batches packaged using transfer plasmids with AAV2-derived ITRs can be quantified with this kit regardless of capsid serotype.
• Precise Quantification Performance: Supplied with DNase I, the kit thoroughly digests residual packaging plasmid contaminants to facilitate accurate absolute copy number quantification of AAV via qPCR for precise dosing of downstream viral applications. Integrated UDG and dUTP further safeguard quantification accuracy by minimizing PCR amplicon carryover artifacts.
• Rapid & Streamlined Workflow: Post-virus harvest, free extraneous nucleic acids are digested with nuclease followed by protein denaturation using lysis reagent per protocol; processed samples are ready for qPCR quantification. The complete analytical workflow finishes within approximately two hours for reliable AAV titer determination.
• Complete Kit Composition for qPCR Assays: The kit includes pre-formulated SYBR Green qPCR Mix (2X, with UDG) and ITR-specific primers. The master mix is pre-mixed with all essential components: BeyoFast™ Taq DNA Polymerase, UDG, PCR buffer, dNTPs, dUTP, SYBR Green I dye, stabilizers and magnesium ions. Primers are designed against conserved AAV2 ITR sequences, enabling ultrasensitive absolute quantification of AAV genomic DNA. A Positive Control template is additionally provided to validate kit functionality and serve as calibrator for standard curve construction.

Average Ct Values for Standard Curve Assay

Features

• Broad Application Scope: This kit is suitable for concentrating lab-produced viral particles including Lentivirus, Retrovirus, Adeno-associated Virus (AAV), Adenovirus and Baculovirus, as well as viruses derived from other eligible sample matrices.
• User-Friendly Operation: Mix the 4× Precipitation Reagent with virus supernatant at a volumetric ratio of 1:3, incubate the mixture at 4 °C for a defined period, then perform centrifugation. Discard the supernatant and resuspend the viral pellet using the kit-supplied Virus Resuspension Buffer.
• Ultracentrifugation-Free Protocol: Lengthy ultrahigh-speed centrifugation is unnecessary. Viral pellets can be readily harvested via conventional refrigerated centrifugation at 3500×g for 0.5–1 hour.
• Superior Virus Concentration Efficiency: Traditional concentration approaches such as ultrafiltration and ultracentrifugation involve lengthy, cumbersome workflows and frequently result in contamination from cellular debris and serum proteins, leading to low virus purity. In contrast, this kit elevates viral titer by 10–100 fold with peak recovery up to ~90%, minimizing virus loss while fully preserving viral biological activity throughout concentration.
• Excellent Post-Concentration Storage Performance: Supplemented with proprietary virus protective reagent, the kit markedly improves the storage stability of concentrated viruses and slows down titer decay during preservation.

Storage

Store the whole kit at −20 °C for a 12‑month shelf life. SYBR Green qPCR Mix (2X, with UDG), Low ROX (50X) and High ROX (50X) must be stored at −20 °C protected from light, and repeated freeze‑thaw cycles should be minimized.

Note

• Perform DNase I digestion strictly in accordance with the user manual; otherwise, residual AAV packaging plasmid interference will lead to overestimated AAV titers.
• Thaw all reagents completely before use and mix gently by inverting the tube, taking care to avoid bubble formation during mixing.
• SYBR Green qPCR Mix (2X, with UDG), Low ROX (50X) and High ROX (50X) contain fluorescent dyes. Protect reagents from intense light during storage and qPCR setup to prevent photobleaching.
• Functional verification confirms no obvious performance deterioration after up to 10 freeze‑thaw cycles; nevertheless, unnecessary repeated freeze‑thawing is still discouraged as prolonged cycling may compromise reagent performance.
• Given the ultrahigh sensitivity of qPCR assays, set up reactions inside a dedicated standard PCR laboratory to minimize amplicon carryover contamination. Despite the kit’s built‑in anti‑carryover UDG system, do not peel sealing films or open PCR tubes in the reaction setup area. All post‑run PCR amplicons must be tightly sealed and disposed of following standard waste regulations to prevent high-concentration amplicon contamination of the workspace.
• Filtered pipette tips are recommended for qPCR master mix preparation to effectively eliminate artificially elevated titer readings caused by contaminant carryover.
• This product is for research use only by qualified laboratory personnel. It shall not be applied for clinical diagnosis, therapeutic treatment, food or pharmaceutical production, nor stored in residential housing.
• Wear a lab coat and disposable gloves throughout all experimental operations for personal safety.

Only for research and not intended for treatment of humans or animals

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SBS Genetech is a long-term sponsor of Cold Spring Harbor Laboratory