User manual

Droplet Digital

™

PCR Tips, Assay Considerations, and Troubleshooting

82 | Droplet Digital

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PCR Applications Guide

Note: The UNG approach can address contamination caused by PCR products created

using ddPCR supermix for probes or one-step RT-ddPCR kit for probes; however, it will not

address contamination caused by sample-source templates or PCR products created using

droplet PCR supermix. Ultramers or long oligo PCR templates are especially problematic

because the stock concentration is typically very high and can easily spread to pipets

and other surfaces. It can be useful to order ultramers with uracils (Us) in place of a few

of the thymidines (Ts) in order to take advantage of UNG treatment in case contamination

becomes a problem.

High Mean Fluorescence Amplitude Intensity

If the ﬂuorescence amplitude of negative droplets is excessively high such that they are all

considered positive and therefore concentration cannot be determined, it is possible the

sample’s target concentration is so high that every droplet contains DNA target and no

negative droplets exist (Figure 9.5). When there are no negative droplets, Poisson correction

cannot be applied and it is not possible to calculate a concentration.

Fig. 9.5. Example of all droplets being positive (containing template) for the FAM assay

and/or the VIC assay.

Channel 1 amplitude

–1,000

1,000

2,000 3,000 4,000

2,000

7,0000

3,000

4,000

5,000 6,000 8,000

Having all positive droplets could arise from multiple issues.

1. Polymerase independent — probe hydrolysis due to poor long-term storage of probe

stock solution, such as in a nonbuffered solution (for example, water) at 4°C.

– Reorder the probe and make probe stock solution with 10 mM Tris, pH 8.0–8.5,

and store at –20°C

2. Polymerase dependent — assay components interact with each other in a way that

results in premature probe cleavage by the enzyme.

– Identify intra-assay interactions and redesign causative component(s) to reduce

binding and cleavage

Note: Run an NTC well to identify this problem. If the negative droplets in the NTC

well do not have high ﬂuorescence amplitude droplets, then the target concentration

is too high. If the negative droplets in the NTC well contain high amplitude droplets, the

cause is either polymerase-independent probe hydrolysis or polymerase-dependent

intra-assay interactions.