You must have a clean working area to titer your AAV by ddPCR. Droplet digital PCR is a sensitive assay, so cross contamination into a no template control (NTC) well is common. Here are some steps you can take to achieve a clean NTC:
- Have a dedicated bench with a dedicated set of pipettes for ddPCR set-up.
- Prepare the master mix in a separate area from where you prepare your sample dilutions.
- Aliquot all of your reagents into single-use tubes and grab a fresh one for each set up.
- Wipe down the bench and all consumables with 10% bleach.
If you are using a manual droplet generator, you will have to be careful about transferring your droplets from the droplet generator to the PCR plate. Maintaining a high droplet count is important for calculating the concentration of your sample.
Even if you are using an automated droplet generator, you should avoid pipetting too quickly when making your virus dilutions. This will reduce aerosols that could potentially lead to contamination.
Optimize your PCR
For a good starting place on what PCR parameters to use, see Lock’s seminal paper on AAV titration (Lock et al., 2014).
If you are having difficulty getting a clean separation between positive and negative droplets with these parameters, here are a few things you can try:
- Increase the number of cycles. After additional rounds of amplification, your fluorescence amplitude will increase, leading to greater separation of your positive and negative droplets. No more than 50 cycles is recommended.
- Decrease your ramp rate. A rate of 2C/s is recommended to ensure an even temperature change among all of the droplets, but you can go as low as 1C/s.
- Increasing the elongation time to 2 minutes and the denaturation time to 1 minute has been shown to increase droplet separation (Witte et al., 2016). This is particularly important if your amplicon is longer than 150 base pairs.
While we use ddPCR for AAV titration, there are many applications for the technology. Droplet digital PCR is well suited for the detection of low copy numbers. For this reason, it can be used in the detection of rare sequences and single cell analysis. It is also being used in the detection of microbes. Some applications include measuring viable probiotics and detecting circulating pathogens.
With its many applications and ease of use, droplet digital PCR is becoming a popular option for PCR experiments.