User manual

14 | Droplet Digital
™
PCR Applications Guide
Fig. 2.2. Main tab in Primer3Plus.
In the Main window, paste your target DNA sequence in the “Paste source sequence
below” field.
We recommend the following changes to the default settings when designing ddPCR assays:
■
In the General Settings window, change “Concentration of divalent cations” to 3.8,
“Concentration of dNTPs” to 0.8, and “Mispriming/Repeat Library” to the correct organism
■
In the Advanced Settings window, change both the “Table of thermodynamic
parameters” and “Salt correction formula” to SantaLucia 1998
■
In the Internal Oligo window, we recommend setting 15 for the minimum number of
bases for the oligo. We recommend 64°C as the minimum T
m
for the probe, 65°C as the
optimal T
m
for the probe, and 70°C as the maximum T
m
for the probe. These parameters
can be relaxed to allow for smaller/larger oligos, which may be necessary for high GC
or low GC targets. Oligo size should be no smaller than 13 and no larger than
30 nucleotides
Note: After you have made the desired changes in Primer3Plus, select Save Settings
under General Settings and save these parameters in a file. To apply these settings in
the future, upload them by selecting Browse in the General Settings tab, find this file,
and click Activate Settings.
After you paste your target sequence into the Main window, click Pick Primers
(Figure 2.3). The software provides one or more primer pairs to select or provides an
explanation for why the software failed to arrive at any primers.
Designing Droplet Digital
™
PCR Experiments