Primer3 : designing primers and probes for ddPCR

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 Tm for the probe, 65°C as the optimal Tm for the probe, and 70°C as the maximum Tm 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.