Syphy-lab · ETH Zürich

AAV Protocol

HEK 293T Large-scale manufacturing — —

📊 Open Lab Data Sheet

Vessel & Scale

Vessel type

15cm Dish (×1)

T500 (×6.36)

HyperflaskM (×13)

Roller Bottle (×14)

CellStack (×23)

Number of vessels

DNA per dish (ng)

Total DNA

—

Equiv. dishes

—

Plasmid Parameters

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Helper

Name

Size (bp)

Conc (ng/µl)

📚 From library

Capsid

Name

Size (bp)

Conc (ng/µl)

📚 From library

Transgene

Name

Size (bp)

Conc (ng/µl)

📚 From library

Molar Ratio Normalisation

Plasmid	Size (bp)	Rel. to Smallest	Fill to 1	ng / dish	pmol / dish

DNA Mix Mastermix

Plasmid	Stock (µg/µl)	Stock (pmol/µl)	µl / dish	µl TOTAL

PEI Mix Mastermix

Component	Per dish (µl)	Total (µl)

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Recipe name / experiment ID *

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📋 Transfection History

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B Buffers & Solutions (click to expand)

5 M NaCl 2 L

584.4 g NaCl (58.44 g/mol) — adjust to 2 L — autoclave

2 M MgCl₂ 100 mL

40.66 g MgCl₂·6H₂O (203.30 g/mol) — adjust to 100 mL — autoclave

2.5 M KCl 100 mL

18.64 g KCl (74.55 g/mol) — adjust to 100 mL — autoclave

AAV Lysis Solution — Benzonase 1 L

50 mL 1M TRIS-HCl (50 mM) | 30 mL 5M NaCl (0.15 M) | 1 mL 2M MgCl₂ (2 mM) | pH 8.5 | autoclave

AAV Lysis Solution — SAN 1 L

50 mL 1M TRIS-HCl pH 8.5 (50 mM) | 200 mL 5M NaCl (1 M) | 5 mL 2M MgCl₂ (10 mM) | pH 8.5 | autoclave

5× AAV Precipitation Buffer 1 L

400 g PEG 8000 (40% w/v) | 146.1 g NaCl (2.5 M) | dissolve PEG by heating to 37 °C (may take overnight)

10× PBS-MK 500 mL

5 packages 1× PBS powder | 2.5 mL 2M MgCl₂ (10 mM) | 5 mL 2.5M KCl (25 mM) | adjust to 500 mL with sterile Milli-Q | do not autoclave

PBS-MK-NaCl 1 L

100 mL 10× PBS-MK | 400 mL 5M NaCl (2 M final) | fill to 1 L with ddH₂O | do not autoclave

AAV Storage Solution 200 mL

180 mL 1× PBS-MK | 20 mL glycerol

⚠ Iodixanol Solutions — prepare in advance, store at 4 °C protected from light

54% Iodixanol stock: 540 mL Optiprep (60%, CAT 1114542) + 60 mL 10× PBS-MK

58% phase: 200 mL Optiprep (60%) + 100 µL 2M MgCl₂ + 200 µL 2.5M KCl + 100 µL 0.5% Phenol Red (turns yellow)

40% phase: 160 mL 54% stock + 56 mL 1× PBS-MK

25% phase: 100 mL 54% stock + 116 mL 1× PBS-MK + 432 µL 0.5% Phenol Red

15% phase: 120 mL 54% stock + 96 mL 1× PBS-MK + 216 mL 1× PBS-MK-NaCl + 650 µL 0.5% Phenol Red

Total 60% Optiprep consumption: ~15.5 mL

Transfection

Day −1 to Day 0

Thaw and expand HEK 293T into 10 T175 flasks, keep cells non-confluent.
Medium: DMEM, 10% FBS, 1% HEPES, no antibiotics.

24 h before transfection, seed cells:

Vessel	Cells to seed	Medium volume
15 cm dish (×23)	15 × 10⁶ per dish	standard
CellStack	250 × 10⁶ total	500 mL / stack
Roller Bottle (2160)	165 × 10⁶ total	standard

Calculate plasmid amounts using the Transfection Calculator tab.
Mix: add NaCl + H₂O first, then plasmid DNA, then PEI.
Vortex 10 s, incubate at RT for 10 min, then add 2 mL / dish (or 46 mL / CellStack).
Wait 72 h without changing medium.

Day 1 — Harvest & Precipitation

72 h post-transfection

⚠ AAVs are stable — clean every spill and surface with 1% bleach.

Harvest supernatant 72 h post-transfection; transfer to 1 L bottle.
Wash vessel with 50 mL PBS and add to the supernatant.
Collect cell pellet:
- CellStack: add 50 mL trypsin, 5 min RT, dislodge cells, collect in falcon, centrifuge 800 × g / 5 min to remove trypsin.
- Dishes: scrape cells and collect in falcon (no trypsin).
Add 5× Precipitation Buffer to supernatant to reach 1×:
PBvol = Svol / 0.8 − Svol (Svol = volume of supernatant)
Invert; incubate at 4 °C for 2 h (or overnight).
Resuspend cell pellet in 6.5 mL AAV Lysis Solution (do not aspirate — pellet is sticky). Store at −80 °C in a proper Falcon tube.
Centrifuge supernatant: 4000 × g, 30 min, 4 °C in large-volume centrifugation bottles.
Resuspend pellet in 6.5 mL AAV Lysis Solution. Store at −80 °C.

Day 2 — Freeze-Thaw & SAN Treatment

Freeze-Thaw Lysis

Thaw cell pellet in 37 °C water bath, then freeze in liquid nitrogen (until bubbling stops).
Repeat for a total of 3 freeze-thaw cycles.
If supernatant contains many detached cells (common), also freeze-thaw the supernatant fraction.
Pool supernatant + cell pellet → ~14.5 mL total AAV solution.

Salt Active Nuclease (SAN) or Benzonase Treatment

Add 50 U SAN per 15 cm² dish (1150 U / CellStack).
Incubate 37 °C, 1 h, 200 rpm shaker (place tube in beaker).
Centrifuge: 4000 × g, 15 min, 4 °C. Collect supernatant. Repeat centrifugation.
Remove floating debris with a serological pipette before loading gradient.

Iodixanol Gradient Setup

Fit QuickSeal tube on holder and insert a 225 mm Pasteur pipette before adding solutions.

Pipetting tips: never touch the Pasteur rim with the stock bottle. Apply P1000 pressure directly over the Pasteur (not dropwise) to prevent hanging at the neck. Keep Pasteur submerged — never let it empty. After AAV solution, weigh tube + pipette and adjust with Lysis Buffer.

Layer (bottom → top)	Solution	Volume
1 (bottom)	58% Iodixanol (yellow)	5 mL
2	40% Iodixanol	5 mL
3	25% Iodixanol (pink)	6 mL
4	15% Iodixanol (pink)	9 mL
5 (top)	AAV solution	14.5 mL
Total		39.5 mL

Weigh full tube + Pasteur. Adjust to match with AAV Lysis Buffer. Tube must be completely full — no air bubbles (tube collapses during spin).
Remove Pasteur while plugging with a finger. Weigh again.
Seal: add metal lid, press with heated plate (not too hard — hot lid must not touch the shoulders). Press with cold plate. Remove lid, squeeze tube gently to verify seal.
Balance tubes in pairs (opposite in rotor). Max 0.01 g difference. Use Tough Spots on caps if needed.
Centrifuge: 63,000 RPM, 2 h, 4 °C (or 69,000 RPM, 1 h 15 min, 4 °C) — Beckman Type 70 Ti rotor.

Collecting Purified AAV

AAV particles accumulate in the 40% Iodixanol phase.

Fix centrifuge tube in bracket stand. Prepare two 15 mL and one 50 mL falcons (uncapped).
Insert a 20G needle at the top of the tube (air vent).
Pierce bottom of tube with a needle, retract, place a 15 mL falcon below. Control flow speed by sealing the top needle with your finger.
Discard initial 4 mL (58% Iodixanol).
Collect following 3.5 mL containing AAV particles. Avoid 25% phase (major protein impurities).
Discard remainder into 50 mL falcon.
Proceed to buffer exchange, or add AAV Storage Solution (up to 15 mL), mix by inversion, store O/N at 4 °C.

Buffer Exchange & Concentration (Amicon 100 kDa)

⚠ All following centrifugation steps MUST occur in a swinging bucket centrifuge with filter perpendicular to motion.

Fill AAV-containing iodixanol fraction to 15 mL with AAV Storage Solution. Invert to mix.
Pre-wet 15 mL Amicon 100 kDa MWCO filter with AAV Storage Solution; centrifuge 800 × g, 5 min, 4 °C.
Load AAV solution into filter. Spin 800 × g, 5 min, 4 °C to concentrate to ~0.5–1 mL.
Discard flow-through.
Refill to 15 mL with AAV Storage Solution. Repeat step 3. Perform 3 wash rounds total.
Concentrate to desired volume: minimum 200 µL, maximum 250 µL.
Tilt falcon, collect concentrated AAV, rinse filter.
Aliquot into 20 µL aliquots in protein low-bind 0.5 mL Eppendorf tubes. Store at −80 °C.
Reserve a 5 µL aliquot for titration by ddPCR.

Experiment Notes

Date

Experimenter

Cell passage

Cell viability %

AAV Titre — ddPCR Protocol

ITR / Transgene

Sample preparation (DNase treatment)

Dilute AAV sample 1:10 in DNase I buffer (10 µl sample + 90 µl buffer).
Add 2 U DNase I; incubate 37 °C, 30 min to digest unpackaged DNA.
Inactivate: 75 °C, 10 min. Cool on ice.
Proteinase K digestion: add 2 µl ProK (20 mg/ml) + SDS to 0.1%. Incubate 56 °C, 30 min.
Dilute 1:100 in nuclease-free H₂O (total dilution 1:1000 from original).

Reaction Setup (per well, 20 µl total)

Component	Stock	µl / rxn
ddPCR Supermix for Probes (no UTP)	2×	10
Forward primer (ITR or transgene)	10 µM	0.9
Reverse primer	10 µM	0.9
Probe (FAM/HEX)	10 µM	0.5
Prepared sample (1:1000 dilution)	—	5
Nuclease-free H₂O	—	2.7
Total		20

Droplet generation & PCR cycling

Load 20 µl reaction + 70 µl Droplet Generation Oil into QX200 cartridge. Generate droplets.
Transfer ~40 µl droplets to PCR plate; seal with pierceable foil at 180 °C, 5 s.

Run on thermocycler (ramp rate 2 °C/s):

Step	Temp	Time	Cycles
Enzyme activation	95 °C	10 min	1
Denaturation	94 °C	30 s	40
Annealing / Extension	60 °C	1 min	40
Signal stabilisation	98 °C	10 min	1
Hold	4 °C	∞	—

Read on QX200 Droplet Reader. Accept wells with >10,000 droplets.
Set threshold between positive and negative droplet populations.
Analysis: titre (GC/ml) = copies/µl × dilution factor × 1000
Account for total dilution from DNase step (1:10) + final dilution (1:100) = 1:1000 minimum.

Primer & Probe References

Target	Sequence (5′→3′)	Tm
ITR-F	`GGAACCCCTAGTGATGGAG`	60 °C
ITR-R	`CGGCCTCAGTGAGCGA`	60 °C
ITR Probe (FAM)	`FAM-CACTCCCTCTCTGCGCGCTCG-BHQ1`	—
Or use transgene-specific primers for insert-based titre.

💧 ddPCR Results & Save

Batch / Sample ID

Date

Serotype

Copies / µl (from reader)

Dilution factor

Accepted droplets

Calculated titre

—

Notes

96-Well Plate Setup Builder

AAVpro® Kit

Samples

Sample reps

Std reps

NTC

ROX

Master Mix

Component	Stock	Per rxn (µl)	Total (µl)

AAVpro® Titration Kit Ver.2 — qPCR Protocol

Takara Cat. #6233

TB Green™-based real-time PCR targeting the AAV2 ITR (works for all serotypes with AAV2-derived ITRs). Primers and positive control are included in the kit.

Vector Genome Extraction (per sample)

DNase I digestion — prepare 20 µl reaction:

Component	µl
AAV particle solution	2
10X DNase I Buffer (kit)	2
DNase I (kit)	1
dH₂O	15
Total	20

Incubate 37 °C, ≥15 min.

Inactivate DNase I: 95 °C, 10 min.
Add 20 µl Lysis Buffer (kit); incubate 70 °C, 10 min.
Dilute ≥50× in EASY Dilution (kit) — use as PCR template.
For typical preps: 1 µl lysate + 99 µl EASY Dilution (100×) is a good starting dilution. Optimise empirically to fall within the standard curve range.

Standard Curve Preparation

Use the kit Positive Control (2 × 10⁷ copies/µl) to make 6-point 1:10 serial dilutions in EASY Dilution:

Point	Copies / µl	Preparation
Std 1	2 × 10⁷	Positive Control (undiluted)
Std 2	2 × 10⁶	5 µl Std 1 + 45 µl EASY Dilution
Std 3	2 × 10⁵	5 µl Std 2 + 45 µl EASY Dilution
Std 4	2 × 10⁴	5 µl Std 3 + 45 µl EASY Dilution
Std 5	2 × 10³	5 µl Std 4 + 45 µl EASY Dilution
Std 6	2 × 10²	5 µl Std 5 + 45 µl EASY Dilution
NTC	—	5 µl dH₂O (no template control)

Prepare 50× Primer Mix (fresh, do not store)

Component	µl
AAV Forward Titer Primer (kit)	5
AAV Reverse Titer Primer (kit)	5
TE buffer or dH₂O	15
Total	25

Reaction Setup (25 µl, on ice)

Prepare mastermix, dispense 20 µl/well, then add 5 µl template.

Component	Stock	µl / rxn (no ROX)	µl / rxn (+ ROX)
TB Green Premix Ex Taq II (kit)	2×	12.5	12.5
50× Primer Mix (freshly prepared)	50×	0.5	0.5
ROX Reference Dye / Dye II	—	—	0.5
dH₂O	—	7.0	6.5
Template (sample or standard)	—	5.0	5.0
Total		25	25

ROX: use ROX Reference Dye for StepOnePlus; ROX Reference Dye II for ABI 7500 Fast. Omit ROX for Takara Thermal Cycler Dice / other non-ABI instruments.

Cycling Conditions

Step	Temp	Time	Cycles
Initial denaturation	95 °C	2 min	1
Denaturation	95 °C	5 s	35
Annealing / Extension (read fluorescence)	60 °C	30 s	35
Melt curve	60 → 95 °C	+0.1 °C/s	1

Analysis & Titre Calculation

Verify standard curve: accept R² > 0.99, efficiency 90–110%. NTC must be negative.
Verify melt curve shows a single peak (no primer dimers).
Interpolate sample Cq → copies/µl from standard curve.
Back-calculate titre accounting for all dilutions:
titre (vg/ml) = copies/µl × dilution factor × 1000
Run each sample in triplicate; report mean ± SD.
The Positive Control is an ITR-containing plasmid quantified by A₂₆₀. It reflects the same ITR copy number as vector genomes, giving absolute vg counts. Targets AAV2 ITR — confirm your vector uses AAV2-derived ITRs.

📈 qPCR Results & Save

Batch / Sample ID

Date

Serotype

Copies / µl (from curve)

Dilution factor

R²

Efficiency %

Calculated titre

—

Notes

Record Titre & Storage

Batch / Experiment ID *

Date *

Titre (vg/µL) *

Method *

Aliquot size (µL) *

Number of Aliquots *

Storage location *

Notes

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Stored Batches

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Concentration (ng/µl) *

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AAV Protocol

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Reagent Checklist

Vessel & Scale

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📋 Transfection History

Transfection

Day 1 — Harvest & Precipitation

Day 2 — Freeze-Thaw & SAN Treatment

Experiment Notes

AAV Titre — ddPCR Protocol

💧 ddPCR Results & Save

96-Well Plate Setup Builder

AAVpro® Titration Kit Ver.2 — qPCR Protocol

📈 qPCR Results & Save

Record Titre & Storage

Stored Batches

🗂 Lab Archive

Add Plasmid to Library

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