AAV Titration

🧬

AAV Titration Calculator

Wet Lab qPCR Protocol  ·  DNAse digestion + serial dilution + plasmid standard ITR qPCR

🧪 1. DNAse Mix per sample

Number of samples (virus preps to titrate)

samples

Total reactions

—

3 dilutions × samples + 1 extra

Total DNAse mix volume

— µL

enough for all reactions

Component	Per Rx (µL)	Total (µL)

💡 Each reaction receives 20 µL DNAse mix. Prepare in PCR hood.

🦠 2. Virus Serial Dilution 1:10 · 1:100 · 1:1000

Stock virus volume (for first dilution)

µL into 20 µL DNAse mix

Transfer volume between dilutions

µL from each tube to next

Tube 1 (1:10)

—

—

Tube 2 (1:100)

—

—

Tube 3 (1:1000)

—

—

—

💡 Mix 20–30 times between transfers. Discard 2.2 µL from last tube.

🧫 3. EDTA & 1:10 Dilution after DNAse inactivation

EDTA volume per tube (500 mM)

µL

Sample volume for 1:10 dilution

µL DNAse-treated sample

Water volume for dilution

µL to make 1:10

Total EDTA needed

— µL

for all tubes

Dilution factor

1:10

sample + water

—

💡 Add EDTA after 37°C incubation, then 75°C 20 min to inactivate DNAse. Then dilute 1:10 to reduce EDTA inhibition.

🧬 4. Positive Control Plasmid plasmid 110 · 9 dilutions

Plasmid stock concentration (µg/µL)

µg/µL

Target concentration for Third dilution (GC/mL)

GC/mL from protocol

Third dilution

— GC/mL

—

—

Dilution	Factor	Concentration (GC/mL)	Volume per well

💡 Prepare on your bench (not in PCR hood) to avoid contaminating master mix. Add 5 µL per well.

📊 5. qPCR Master Mix PerfeCTa 2× SuperMix

Number of virus samples

samples × 3 dilutions

Number of plasmid controls (including negative)

plasmid dilutions

Negative control wells

water only

Total wells

—

virus + plasmid + neg

Total master mix

— µL

with overage

Component	Per Well (µL)	Total (µL)

💡 Add 5 µL master mix per well, then 5 µL sample. Total 10 µL per well. Prepare in PCR hood.

⏱️ AAV Titration Protocol – Quick Reference

1DNAse mix: Prepare master mix (calc above) in PCR hood.~5 min

2Serial dilution: Add 20 µL DNAse mix + 2.2 µL virus (1:10), transfer 2.2 µL to 1:100, then 1:1000.~10 min

3DNAse digest: 37°C for 30 min, then 4°C hold.30 min

4EDTA + heat: Add 1 µL 500 mM EDTA, 75°C for 20 min.20 min

51:10 dilution: 5 µL sample + 45 µL water to dilute EDTA.~5 min

6Plasmid controls: Prepare serial dilutions on bench (calc above).~20 min

7qPCR master mix: Prepare in PCR hood (calc above), add 5 µL to plate.~10 min

8Load samples: Add 5 µL per well in virus hood. Total 10 µL/well.~15 min

9Run qPCR: 50°C 60s → 95°C 120s → [95°C 15s, 60°C 60s] × 40 cycles.~90 min

💡 Critical: Keep virus in virus hood, plasmid on bench, master mix in PCR hood to avoid cross‑contamination.

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