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

[DESCRIPTION]
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.

[GUIDELINES]
This protocol was modified from the publication Lock M, Alvira MR, Chen SJ, Wilson, 2014.
The dilution series outlined in this protocol is based on an AAV titer range of 5E+12 - 5E+13 GC/mL. Users may need to run lower or higher dilutions depending on their particular sample.

[STEPS]
SECTION: Preparation
1. Before handling any viruses get materials ready.
SECTION: Preparation
2. Ensure that primers, probe, 10X PCR buffer, and master mix are thawed.
SECTION: Preparation
3. Vortex primers, probe and master mix for 15 s then spin 10 s in a mini centrifuge and place on ice.

SECTION: Preparation
4. Wipe down a DG8 cartridge holder with bleach and place in the Biological Safety Cabinet (BSC).
SECTION: Preparation
5. 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.
 

SECTION: Preparation
6. Pre-warm the 96-well plate sealer by gently touching the screen.
SECTION: Prepare the Serial Dilution
7. Place a 48-well dilution plate in a chilled 96-well freezer block and place in the dilution BSC.
SECTION: Prepare the Serial Dilution
8. Prepare 1X dilution buffer (see recipe in reagent section).
SECTION: Prepare the Serial Dilution
9. Pour the 1X dilution buffer into a polystyrene reagent reservoir.
SECTION: Prepare the Serial Dilution
10. Using a 20-200µl multichannel pipette, carefully add the dilution buffer to the dilution plate according to the dilution scheme listed in step 12.
SECTION: Prepare the Serial Dilution
11. Use a single channel 1-10µl pipette to add 5 µL of each viral sample to Dilution 1 in the 48-well dilution plate and pipette 5-10 times to mix.

SECTION: Prepare the Serial Dilution
12. Dilute the virus as follows: Mix dilutions thoroughly, but pipette slowly to avoid generating aerosols. 

Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer (1:20) 
Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer (1:400) 
Dilution 3 (20X): 5 µL in 95 µL 1X PCR buffer (1:8,000) 
Dilution 4 (20X): 5 µL in 95 µL 1X PCR buffer (1:160,000) 
Dilution 5 (20X): 5 µL in 95 µL 1X PCR buffer (1:3,200,000) 
Dilution 6 (2X): 50 µL in 50 µL 1X PCR buffer (1:6,400,000) 
Dilution 7 (2X): 50 µL in 50 µL 1X PCR buffer (1:12,800,000) 
Dilution 8 (2x): 50 µL in 50 µL 1X PCR buffer (1:25,600,000)
SECTION: Prepare the Serial Dilution
13. Use multichannel pipettes for the dilution series.
 

SECTION: Prepare the Serial Dilution
14. Gently cover the entire dilution plate with Microseal adhesive seal - do not press the film just gently cover so nothing falls into the plate.
SECTION: Prepare the Serial Dilution
15. Leave dilutions on the chilled 96-well freezer block in the BSC until ready to use.
SECTION: Prepare the Master Mix
16. Place a ddPCR plate onto a chilled 96-well freezer block and set aside in the droplet generation BSC to cool.
SECTION: Prepare the Master Mix
17. Prepare the ITR master mix in a microcentrifuge tube as shown below. For 8 samples prepare enough master mix for 9 samples.  
 

SECTION: Prepare the Master Mix
18. Vortex the master mix for 15 s and spin in a mini centrifuge for 10 s before use.
SECTION: Prepare the Master Mix
19. Place an 8-well PCR tube strip into a chilled 96-well freezer block.
SECTION: Prepare the Master Mix
20. Add 20 µL 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.

SECTION: Prepare the Master Mix
21. Cap gently - do not push the cap in all the way, just ensure the samples are covered.
SECTION: Prepare the Master Mix
22. Bring the PCR tubes to the BSC used for dilutions.
SECTION: Prepare the Master Mix
23. Without disturbing the liquid in the tube, gently uncap the PCR tubes.
SECTION: Prepare the Master Mix
24. Add 5 µL of dilutions 6-8 to the appropriate PCR tubes. Pipette back and forth 5 times.

SECTION: Prepare the Master Mix
25. Lightly cap the PCR tubes.
SECTION: Generate the Droplets
26. Bring the PCR tubes to the droplet generation BSC.
SECTION: Generate the Droplets
27. Without disturbing the liquid in the tube, gently uncap the tubes.
SECTION: Generate the Droplets
28. Add 5 µL nuclease-free water to the NTC tube.

SECTION: Generate the Droplets
29. Place a DG8 cartridge into the cartridge holder.
SECTION: Generate the Droplets
30. Using a 2-50µL multichannel pipet, load 20 µL of the reaction mixtures into the middle wells of the cartridge.

SECTION: Generate the Droplets
31. Add 800 µL of droplet generation oil to a polystyrene reagent reservoir.

SECTION: Generate the Droplets
32. Using the 20-200µL multichannel pipet, load 70 µL of droplet generation oil into the bottom row of wells.

SECTION: Generate the Droplets
33. Cover the cartridge with the DG8 gasket, making sure that it is secure.
SECTION: Generate the Droplets
34. Transfer the cartridge holder to the droplet generator. Close the lid and wait for the droplets to be generated.
SECTION: Generate the Droplets
35. Once the droplets have been generated, use a 20-200µL multichannel pipet to aspirate 40 µL  of droplets. 
 

SECTION: Generate the Droplets
36. Transfer the droplets to a prechilled PCR plate.
 

SECTION: Generate the Droplets
37. 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.
SECTION: Generate the Droplets
38. After the plate has been sealed, proceed to thermocycling.
SECTION: Thermal Cycling
39. Run the following PCR parameters.
 

SECTION: Thermal Cycling
40. After PCR is complete, transfer the plate to the Droplet Reader.
SECTION: Thermal Cycling
41. 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.
SECTION: Thermal Cycling
42. When the plate layout is complete, select 'Run' to begin the droplet reading.
SECTION: Thermal Cycling
43. 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