TITLE: ddPCR titration of AAV vectors
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes ddPCR titration of AAV vectors. To see the full abstract and additional resources, visit </span><a href="https://www.addgene.org/protocols/aav-ddpcr-titration" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/aav-ddpcr-titration/</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Sample Data: </span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">When analyzing data there should be a clear distinction between negative droplets (black) and positive droplets (blue).</li><li style = "counter-reset:ol0;list-style-type:disc;">The no template control (NTC) should be close to zero (B08). At Addgene, runs with an NTC >5 are invalid.</li><li style = "counter-reset:ol0;list-style-type:disc;">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.</li><li style = "counter-reset:ol0;list-style-type:disc;">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. </li><li style = "counter-reset:ol0;list-style-type:disc;">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.</li><li style = "counter-reset:ol0;list-style-type:disc;">To increase the accuracy of the titer, calculate an average of several dilutions.</li></ul></div><div class = "text-block"><div class = "justify" style = "text-align:center"><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;"> </span></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/5acd19e056bdf88808866a63dd76bebaf3bfacef4acb2f29cfb4ece3805005bf/b39z7esn.png" /></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;"> </span></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/5acd19e056bdf88808866a63dd76bebaf3bfacef4acb2f29cfb4ece3805005bf/b3927esn.png" /></div><div class = "text-block"><div class = "justify" style = "text-align:center"><a href="https://blog.addgene.org/droplet-digital-pcr-for-aav-quantitation" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;">For additional tips on AAV titering using ddPCR, read our blog post.</span></a></div></div></div>

[STEPS]
?. [Preparation]
Before handling any viruses get materials ready.
?. [Preparation]
Ensure that primers, probe, 10X PCR buffer, and master mix are thawed.
?. [Preparation]
Vortex primers, probe and master mix for  then spin  in a mini centrifuge and place .
on ice
?. [Preparation]
Wipe down a DG8 cartridge holder with bleach and place in the Biological Safety Cabinet (BSC).
?. [Preparation]
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.
*Pro-Tip*Prepare the viral dilution in a separate biological safety cabinet (dilution BSC) than that used for master mix preparation and droplet generation (droplet generation BSC).
?. [Preparation]
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 the Serial Dilution]
Prepare 1X dilution buffer (see recipe in reagent section).
?. [Prepare the Serial Dilution]
Pour the 1X dilution buffer into a polystyrene reagent reservoir.
?. [Prepare the Serial Dilution]
Using a 20-200µl multichannel pipette, carefully add the dilution buffer to the dilution plate according to the dilution scheme listed in step 12.
?. [Prepare the Serial Dilution]
Use a single channel 1-10µl pipette to add  of each viral sample to Dilution 1 in the 48-well dilution plate and pipette 5-10 times to mix.
5 µl
?. [Prepare the Serial Dilution]
Dilute the virus as follows: Mix dilutions thoroughly, but pipette slowly to avoid generating aerosols. Dilution 1 (20X):  in  H2O (1:20) Dilution 2 (20X):  in  H2O (1:400) Dilution 3 (20X):  in  H2O (1:8,000) Dilution 4 (20X):  in  H2O (1:160,000) Dilution 5 (20X):  in  H2O (1:3,200,000) Dilution 6 (2X):  in  H2O (1:6,400,000) Dilution 7 (2X):  in  H2O (1:12,800,000) Dilution 8 (2x):  in  H2O (1:25,600,000)
5 µl
95 µl
5 µl
95 µl
5 µl
95 µl
5 µl
95 µl
5 µl
95 µl
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
?. [Prepare the Serial Dilution]
Use multichannel pipettes for the dilution series.
For dilutions 1-5, use the 1-10µL multichannel pipette set to  .For dilutions 6-8, use the 20-200µL multichannel pipette set to  .For mixing between dilutions, use the 20-200µL multichannel pipette set to   and mix by pipetting the liquid up and down 10-20 times.
?. [Prepare the Serial Dilution]
Gently cover the entire dilution plate with Microseal adhesive seal - do not press the film just gently cover so nothing falls into the plate.
?. [Prepare the Serial Dilution]
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 Master Mix]
Prepare the ITR master mix in a microcentrifuge tube as shown below. For 8 samples prepare enough master mix for 9 samples.    ABCD1ITR Master MixVolume9X VolumeFinal Concentration22X ddPCR Supermix for Probes, no dUTP10µL90µL1X310uM ITR probe (FAM)0.5µL4.5µL250nM4Forward ITR Primer (10uM)1.8µL16.2µL900nM5Reverse ITR Primer (10uM)1.8µL16.2µL900nM6Nuclease-free water5.9µL71.55µL
7Total Volume20µL
ABCD1ITR Master MixVolume9X VolumeFinal Concentration22X ddPCR Supermix for Probes, no dUTP10µL90µL1X310uM ITR probe (FAM)0.5µL4.5µL250nM4Forward ITR Primer (10uM)1.8µL16.2µL900nM5Reverse ITR Primer (10uM)1.8µL16.2µL900nM6Nuclease-free water5.9µL71.55µL
7Total Volume20µL
?. [Prepare the Master Mix]
Vortex the master mix for  and spin in a mini centrifuge for  before use.
?. [Prepare the Master Mix]
Place an 8-well PCR tube strip into a chilled 96-well freezer block.
?. [Prepare the Master Mix]
Add  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.
20 µl
?. [Prepare the Master Mix]
Cap gently - do not push the cap in all the way, just ensure the samples are covered.
?. [Prepare the Master Mix]
Bring the PCR tubes to the BSC used for dilutions.
?. [Prepare the Master Mix]
Without disturbing the liquid in the tube, gently uncap the PCR tubes.
?. [Prepare the Master Mix]
Add  of dilutions 6-8 to the appropriate PCR tubes. Pipette back and forth 5 times.
5 µl
?. [Prepare the Master Mix]
Lightly cap the PCR tubes.
?. [Generate the Droplets]
Bring the PCR tubes to the droplet generation BSC.
?. [Generate the Droplets]
Without disturbing the liquid in the tube, gently uncap the tubes.
?. [Generate the Droplets]
Add  nuclease-free water to the NTC tube.
5 µl
?. [Generate the Droplets]
Place a DG8 cartridge into the cartridge holder.
?. [Generate the Droplets]
Using a 2-50µL multichannel pipet, load  of the reaction mixtures into the middle wells of the cartridge.
20 µl
?. [Generate the Droplets]
Add  of droplet generation oil to a polystyrene reagent reservoir.
800 µl
?. [Generate the Droplets]
Using the 20-200µL multichannel pipet, load  of droplet generation oil into the bottom row of wells.
70 µl
?. [Generate the Droplets]
Cover the cartridge with the DG8 gasket, making sure that it is secure.
?. [Generate the Droplets]
Transfer the cartridge holder to the droplet generator. Close the lid and wait for the droplets to be generated.
?. [Generate the Droplets]
Once the droplets have been generated, use a 20-200µL multichannel pipet to aspirate   of droplets.
40 µl
*Pro-Tip* To ensure that the droplets are not disrupted insert the pipette tips directly in the center of the well and tilt to a 45o angle. Count to 20 while slowly and gently aspirating the droplets.
?. [Generate the Droplets]
Transfer the droplets to a prechilled PCR plate.
*Pro-Tip* To ensure that the droplets are not disrupted insert the pipette tips and gently touch the bottom of the well. Lift the tips ~1mm. Touch the side of the well and tilt the pipette tips at a 45o angle. Count to 20 while slowly and gently dispensing the droplets down the side of the tube.
?. [Generate the Droplets]
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.
?. [Generate the Droplets]
After the plate has been sealed, proceed to thermocycling.
?. [Thermal Cycling]
Run the following PCR parameters.  ABCDE1Cycling StepTemperature (deg C)Time (min)Ramp Rate (deg C/sec)# Cycles2Denaturation9510213Denaturation950.52504Annealing/Extension6012505Signal Stabilization9810216Hold4∞21
ABCDE1Cycling StepTemperature (deg C)Time (min)Ramp Rate (deg C/sec)# Cycles2Denaturation9510213Denaturation950.52504Annealing/Extension6012505Signal Stabilization9810216Hold4∞21
?. [Thermal Cycling]
After PCR is complete, transfer the plate to the Droplet Reader.
?. [Thermal Cycling]
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.
?. [Thermal Cycling]
When the plate layout is complete, select 'Run' to begin the droplet reading.
?. [Thermal Cycling]
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