User manual

Droplet Digital
™
PCR Applications Guide | 81
Droplet Digital
™
PCR Tips, Assay Considerations, and Troubleshooting
Positive Droplets in No Template Control Wells
Digital PCR can detect very low levels of target DNA so it is important to prevent template/
amplicon contamination and to run no template controls (NTCs). Positive droplets in NTC
wells that are at intensities equal to those of positive droplets in sample wells are typically
caused by template or PCR product (amplicon) contamination in the reagents. Having a
clean environment and clean NTC wells (that is, no positive droplets) is imperative when the
application is rare sequence detection (wells with a low number of positives). In Figure 9.4,
the first well (a contaminated NTC well) has four droplets of the same amplitude as those
seen in the well on the right (positive sample).
If positive droplets in NTC wells occur, make sure that good laboratory practices for PCR
are being followed in the laboratory (Kwok and Higuchi 1989).
Suggested guidelines are as follows:
■
Wipe down pipets, tip boxes, and benchtops with 5–10% bleach
■
Prepare master mixes in a template-free environment, add samples and generate
droplets in an amplicon-free environment, perform PCR, and read droplets in a room
separate from the sample preparations
■
Do not reuse DG8
™
droplet generator cartridges, oils, gaskets, plates, or pipet tips
■
Wear appropriate personal protective equipment that is discarded or confined to
appropriate locations (that is, template-free room for master mix assembly, amplicon-free
room for template addition and droplet generation, and PCR and post-PCR rooms for
droplet reading)
Fig. 9.4. An example of contamination of an NTC well (left) with amplitudes similar to a positive template
reaction (right).
Channel 2 amplitude
0
5,000
5,000
1,000
Event number
10,000 15,000 20,000
9,000
2,000
8,000
25,0000
D01 D02
3,000
4,000
6,000
7,000
If desired, dUTP-containing supermixes (ddPCR supermix for probes) can be used in
conjunction with heat-labile uracil N-glycosylase (UNG) or uracil DNA glycosylase (UDG) to
reduce the potential for false positives resulting from the presence of previously amplified
products. Add the UNG at 0.05 units/20 µl of ddPCR reaction mixture and create droplets
as normal. Transfer the foil-sealed 96-well PCR plate containing droplets to the thermal
cycler and add a 30 min 37°C incubation step in front of the standard recommended
thermal cycling protocol. During this incubation period, UNG will digest U-containing
products, such as those from past experiments performed with the ddPCR supermix for
probes. UNG is heat inactivated during the first 3–10 min of the 95°C initial PCR hot-start
step in the standard PCR protocol.