TITLE: ddPCR Titration of Lentivirus Vectors
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes ddPCR titration of Lentivirus vectors. To see the full abstract and additional resources, visit </span><a href="https://www.addgene.org/protocols/lentivirus-ddpcr-titration" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/lentivirus-ddpcr-titration/</span></a><span style = ":;">.</span></div><div class = "text-block"><span>This protocol was modified from the publication </span><a href="https://www.ncbi.nlm.nih.gov/pubmed/29378428" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">Wang Y, Bergelson S, Feschenko M, 2018</span></a><span>. </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/green).</li><li style = "counter-reset:ol0;list-style-type:disc;">The concentration of RRE positive droplets in the untransduced control should be close to zero (A01).</li><li style = "counter-reset:ol0;list-style-type:disc;">In this protocol, the lentiviral particles are serially diluted and used to transduce HEK293T cells. Genomic DNA is extracted from the target cells and assayed for integrated copies of RRE. Since the samples that are assayed are diluted 2-fold serially, the concentration of RRE positive droplets should decrease by a factor of 2 across the dilutions. RPP30 copies should be relatively constant across samples.</li><li style = "counter-reset:ol0;list-style-type:disc;">In the RRE example below, 2-fold serial dilutions of a sample were loaded in wells B01-H01.</li><li style = "counter-reset:ol0;list-style-type:disc;">As shown in the image and table below, the concentration of RRE positive droplets increases by a factor of ~2 as you progress from the higher dilutions to the lower dilutions (blue).</li><li style = "counter-reset:ol0;list-style-type:disc;">The concentration of RPP30 positive droplets stays relatively even across samples (green).</li><li style = "counter-reset:ol0;list-style-type:disc;">To increase the accuracy of the titer, calculate and 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/b5u27esn.png" /></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/b5u37esn.png" /></div><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = ":;"> </span><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/b5u57esn.png" /></div></div>

[STEPS]
?. [Transducing Cells]
Thaw the virus.
on ice
?. [Transducing Cells]
Prepare the following dilution series of virus in DMEM complete:   ABCD1Dilution FactorVirusMediaTotal Volume22.5160µL viral stock240µL400µL35200µL of 2.5-fold dilution200µL400µL410200µL of 5-fold dilution 200µL400µL520200µL of 10-fold dilution200µL400µL640200µL of 40-fold dilution200µL400µL780200µL of 80-fold dilution200µL400µL8160200µL of 160-fold dilution200µL400µL
When adding the virus to the diluent, pipette 5 times, to remove virus from pipette tip. To mix each dilution, set the P200 pipette to   and pipette 10-20 times. Use a new pipette tip to transfer the virus to the next dilution. Repeat for all dilutions.
ABCD1Dilution FactorVirusMediaTotal Volume22.5160µL viral stock240µL400µL35200µL of 2.5-fold dilution200µL400µL410200µL of 5-fold dilution 200µL400µL520200µL of 10-fold dilution200µL400µL640200µL of 40-fold dilution200µL400µL780200µL of 80-fold dilution200µL400µL8160200µL of 160-fold dilution200µL400µL
?. [Transducing Cells]
Add  of each viral dilution to a well of a 6-well plate (1 dilution per well). Leave one well untransduced.
150 µl
?. [Transducing Cells]
Seed 300,000 cells/well in  media and 11.1µg/mL Polybrene into the wells containing the virus and the untransduced control.
1350 µl
*Pro-Tip* For even seeding, prepare a batch for 10 wells with 3,000,000 cells in 13.5 mL of media and 11.1µg/mL Polybrene. Mix the cell suspension well before seeding.
When seeding,   of virus is being diluted an additional 10-fold in media therefore the final dilutions range from 25-1600-fold.
?. [Transducing Cells]
Mix each well with a 1mL pipette 5-10 times.
The final volume in the well is 1.5mL and the final Polybrene concentration is 10µg/mL.
?. [Transducing Cells]
Mix well and incubate .
?. [Benzonase Digestion and Cell Harvest]
Gently aspirate media from the 6-well plates.
?. [Benzonase Digestion and Cell Harvest]
Add  of 50U/mL Benzonase in DMEM complete to each well.
1.5 mL
?. [Benzonase Digestion and Cell Harvest]
Incubate at  for .
37 °C
?. [Benzonase Digestion and Cell Harvest]
Gently aspirate media from wells.
?. [Benzonase Digestion and Cell Harvest]
Wash wells with  of 1X PBS and aspirate.
1 mL
?. [Benzonase Digestion and Cell Harvest]
Detach cells by incubating with  TrypLE for -.
200 µl
?. [Benzonase Digestion and Cell Harvest]
Resuspend cells in  DMEM complete and transfer to a microcentrifuge tube.
500 µl
?. [Benzonase Digestion and Cell Harvest]
Centrifuge for  at .
Centrifuge: 100 34
?. [Benzonase Digestion and Cell Harvest]
Gently aspirate supernatant.
?. [Benzonase Digestion and Cell Harvest]
Wash cell pellets in  PBS.
500 µl
?. [Benzonase Digestion and Cell Harvest]
Centrifuge for  at .
Centrifuge: 100 34
?. [Benzonase Digestion and Cell Harvest]
Gently aspirate supernatant.
?. [Benzonase Digestion and Cell Harvest]
Extract genomic DNA according to the GeneJet Genomic DNA Purification Kit instructions.
?. [Benzonase Digestion and Cell Harvest]
Determine the concentration of each sample on a spectrophotometer.
?. [Benzonase Digestion and Cell Harvest]
Prepare 25ng/µL stocks of each sample. Samples can be used for ddPCR immediately or stored at  until ready to use.
-20 °C
?. [Preparation for ddPCR]
Thaw samples, primers/probe mixes, and master mix on ice.
?. [Preparation for ddPCR]
Before handling any viruses get materials ready.
?. [Preparation for ddPCR]
Vortex primers/probe mixes and master mix for  then spin  in a mini centrifuge and place on ice.
?. [Preparation for ddPCR]
Wipe down a DG8 cartridge holder with bleach and place in the Biological Safety Cabinet (BSC).
?. [Preparation for ddPCR]
Make sure that the BSC is supplied with sufficient pipette tips.
?. [Preparation for ddPCR]
Pre-warm the 96-well plate sealer by gently touching the screen.
?. [Preparation of the Master Mix]
Place a ddPCR plate onto a chilled 96-well freezer block and set aside in the BSC to cool.
?. [Preparation of the Master Mix]
Prepare the RRE/RPP30 master mix in a microcentrifuge tube as shown below. For 8 samples prepare enough master mix for 9 samples.  ABCD1ComponentVolume9X VolumeFinal Concentration22X ddPCR Supermix for Probes, no dUTP10µL90µL1X320X RRE target primers/probe (FAM)1µL9µL900nM/250nM420X RPP30 primers/probe (HEX/VIC) 1µL9µL900nM/250nM5Nuclease-free water4µL36µL
6Total Volume16µL144µL
ABCD1ComponentVolume9X VolumeFinal Concentration22X ddPCR Supermix for Probes, no dUTP10µL90µL1X320X RRE target primers/probe (FAM)1µL9µL900nM/250nM420X RPP30 primers/probe (HEX/VIC) 1µL9µL900nM/250nM5Nuclease-free water4µL36µL
6Total Volume16µL144µL
?. [Preparation of the Master Mix]
Vortex the master mix for  and spin in a mini centrifuge for  before use.
?. [Preparation of the Master Mix]
Place an 8-well PCR tube strip into a chilled 96-well freezer block.
?. [Preparation of 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.
16 µl
?. [Preparation of the Master Mix]
Add  of the 25ng/µL samples the appropriate PCR tubes. Pipette back and forth 5 times.
4 µl
?. [Generating the Droplets]
Place a DG8 cartridge into the cartridge holder.
?. [Generating the Droplets]
Using a 2-50µL multichannel pipet, load  of the reaction mixtures into the middle wells of the cartridge.
20 µl
?. [Generating the Droplets]
Add  of droplet generation oil to a polystyrene reagent reservoir.
800 µl
?. [Generating the Droplets]
Using the 20-200µL multichannel pipet, load  of droplet generation oil into the bottom row of wells.
70 µl
?. [Generating the Droplets]
Cover the cartridge with the DG8 gasket, making sure that it is secure.
?. [Generating the Droplets]
Transfer the cartridge holder to the droplet generator. Close the lid and wait for the droplets to be generated.
?. [Generating the Droplets]
Once the droplets have been generated, use a 20-200µL multichannel pipette 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 45-degree angle. Count to 20 while slowly and gently aspirating the droplets.
?. [Generating 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 45-degree angle. Count to 20 while slowly and gently dispensing the droplets down the side of the tube.
?. [Generating 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.
?. [Generating the Droplets]
After the plate has been sealed, proceed to thermocycling.
?. [Thermal Cycling]
Run the following PCR parameters:  ABCDE1Cycling StepTemperature (oC)Time (min)Ramp Rate (oC/sec)# Cycles2Enzyme Activation9510213Denaturation940.52404Annealing/Extension6012405Enzyme Deactivation9810216Hold4∞21
ABCDE1Cycling StepTemperature (oC)Time (min)Ramp Rate (oC/sec)# Cycles2Enzyme Activation9510213Denaturation940.52404Annealing/Extension6012405Enzyme Deactivation9810216Hold4∞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.
?. [Calculations]
To calculate the titers, first calculate the number of viruses per genome: Viruses/genome = 2*(Copies/20µL RRE)/(Copies/20µL RPP30)
?. [Calculations]
Use the viruses/genome to calculate the infectious titer: Infectious titer = (viruses/genome)*(#cells/well)*(dilution factor)/(virus volume)
?. [Calculations]
Take the average infectious titer obtained from the appropriate dilutions to calculate the final infectious titer.