ddPCR titration of AAV vectors
Addgene The Nonprofit Plasmid Repository
Abstract
This protocol describes ddPCR titration of AAV vectors. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/aav-ddpcr-titration/.
Sample Data:
- When analyzing data there should be a clear distinction between negative droplets (black) and positive droplets (blue).
- The no template control (NTC) should be close to zero (B08). At Addgene, runs with an NTC >5 are invalid.
- To reduce NTC values, we recommend wiping down all pipettes and equipment with 10% bleach prior to use and keeping all reagents and samples on ice or pre-chilled 96-well freezer blocks during use.
- In this protocol, a dilution series is prepared for each AAV sample and the 3 final dilutions are assayed. The samples that are assayed are diluted 2-fold serially therefore, the concentration obtained by ddPCR should decrease by a factor of 2 across the dilutions.
- In the example below, 2-fold serial dilutions of a sample were loaded in wells A04, A05 and A06. As shown in the image and table below, the concentration of positive droplets decreases by a factor of ~2.
- To increase the accuracy of the titer, calculate an average of several dilutions.
For additional tips on AAV titering using ddPCR, read our blog post.
Before start
To reduce the risk of contaminating reagents we recommend making small aliquots of master mixes, primers and probes prior to use.* Thaw the master mix, primers, and probe on ice before use.
- Wipe down all pipettes and surfaces with 10% bleach.
Steps
Preparation
Before handling any viruses get materials ready.
Ensure that primers, probe, 10X PCR buffer, and master mix are thawed.
Vortex primers, probe and master mix for 0h 0m 15s then spin 0h 0m 10s in a mini centrifuge and place On ice.
Wipe down a DG8 cartridge holder with bleach and place in the Biological Safety Cabinet (BSC).
Make sure that the BSC to be used for dilution and the BSC to be used for droplet generation are supplied with sufficient pipette tips and reagent reservoirs.
Pre-warm the 96-well plate sealer by gently touching the screen.
Prepare the Serial Dilution
Place a 48-well dilution plate in a chilled 96-well freezer block and place in the dilution BSC.
Prepare 1X dilution buffer (see recipe in reagent section).
Pour the 1X dilution buffer into a polystyrene reagent reservoir.
Using a 20-200µl multichannel pipette, carefully add the dilution buffer to the dilution plate according to the dilution scheme listed in step 12.
Use a single channel 1-10µl pipette to add 5µL of each viral sample to Dilution 1 in the 48-well dilution plate and pipette 5-10 times to mix.
Dilute the virus as follows: Mix dilutions thoroughly, but pipette slowly to avoid generating aerosols.
- Dilution 1 (20X):
5µLin95µL1X PCR buffer (1:20) - Dilution 2 (20X):
5µLin95µL1X PCR buffer (1:400) - Dilution 3 (20X):
5µLin95µL1X PCR buffer (1:8,000) - Dilution 4 (20X):
5µLin95µL1X PCR buffer (1:160,000) - Dilution 5 (20X):
5µLin95µL1X PCR buffer (1:3,200,000) - Dilution 6 (2X):
50µLin50µL1X PCR buffer (1:6,400,000) - Dilution 7 (2X):
50µLin50µL1X PCR buffer (1:12,800,000) - Dilution 8 (2x):
50µLin50µL1X PCR buffer (1:25,600,000)
Use multichannel pipettes for the dilution series.
Gently cover the entire dilution plate with Microseal adhesive seal - do not press the film just gently cover so nothing falls into the plate.
Leave dilutions on the chilled 96-well freezer block in the BSC until ready to use.
Prepare the Master Mix
Place a ddPCR plate onto a chilled 96-well freezer block and set aside in the droplet generation BSC to cool.
Prepare the ITR master mix in a microcentrifuge tube as shown below. For 8 samples prepare enough master mix for 9 samples.
| A | B | C | D |
|---|---|---|---|
| ITR Master Mix | Volume | 9X Volume | Final Concentration |
| 2X ddPCR Supermix for Probes, no dUTP | 10µL | 90µL | 1X |
| 10uM ITR probe (FAM) | 0.5µL | 4.5µL | 250nM |
| Forward ITR Primer (10uM) | 1.8µL | 16.2µL | 900nM |
| Reverse ITR Primer (10uM) | 1.8µL | 16.2µL | 900nM |
| Nuclease-free water | 5.9µL | 71.55µL | |
| Total Volume | 20µL |
Table: ITR Master mix for 9 samples
Vortex the master mix for 0h 0m 15s and spin in a mini centrifuge for 0h 0m 10s before use.
Place an 8-well PCR tube strip into a chilled 96-well freezer block.
Add 20µL of the master mix to each PCR tube. Be careful to dispense to the bottom of the tube without collecting drops along the side of the tube.
Cap gently - do not push the cap in all the way, just ensure the samples are covered.
Bring the PCR tubes to the BSC used for dilutions.
Without disturbing the liquid in the tube, gently uncap the PCR tubes.
Add 5µL of dilutions 6-8 to the appropriate PCR tubes. Pipette back and forth 5 times.
Lightly cap the PCR tubes.
Generate the Droplets
Bring the PCR tubes to the droplet generation BSC.
Without disturbing the liquid in the tube, gently uncap the tubes.
Add 5µL nuclease-free water to the NTC tube.
Place a DG8 cartridge into the cartridge holder.
Using a 2-50µL multichannel pipet, load 20µL of the reaction mixtures into the middle wells of the cartridge.
Add 800µL of droplet generation oil to a polystyrene reagent reservoir.
Using the 20-200µL multichannel pipet, load 70µL of droplet generation oil into the bottom row of wells.
Cover the cartridge with the DG8 gasket, making sure that it is secure.
Transfer the cartridge holder to the droplet generator. Close the lid and wait for the droplets to be generated.
Once the droplets have been generated, use a 20-200µL multichannel pipet to aspirate 40µL of droplets.
Transfer the droplets to a prechilled PCR plate.
Place a Pierceable Foil Heat Seal on the PCR plate with the red line facing up. If the plate sealer is not at temperature, touch the screen on the plate sealer to allow it to get to temperature. Once the temperature is reached, place the PCR plate with the foil onto the metal support block. Place the block in the plate sealer and press the ‘Seal’ button.
After the plate has been sealed, proceed to thermocycling.
Thermal Cycling
Run the following PCR parameters.
| A | B | C | D | E |
|---|---|---|---|---|
| Cycling Step | Temperature (deg C) | Time (min) | Ramp Rate (deg C/sec) | # Cycles |
| Denaturation | 95 | 10 | 2 | 1 |
| Denaturation | 95 | 0.5 | 2 | 50 |
| Annealing/Extension | 60 | 1 | 2 | 50 |
| Signal Stabilization | 98 | 10 | 2 | 1 |
| Hold | 4 | ∞ | 2 | 1 |
Table: ddPCR parameters
After PCR is complete, transfer the plate to the Droplet Reader.
Open the QuantaSoft software to set up a new plate layout. Designate the sample name, experiment type, supermix type (ddPCR Supermix for Probes), the target names and target types.
When the plate layout is complete, select 'Run' to begin the droplet reading.
When the droplet reading is complete, export the data from all wells as a CSV file which will be used to calculate the titer.
T = {[(R*C)(1000/V)]*D}
T = GC/mL R = reaction volume (20µL)
C = Copies/µL
V = volume of virus in reaction mix (4µL)
D = Dilution factor of virus
