Multiplex primer prediction software for divergent targets

Gardner, Shea N.; Hiddessen, Amy L.; Williams, Peter L.; Hara, Christine; Wagner, Mark C.; Colston, Bill W.

For confirmation of amplicon sequence, 9 ml of amplified PCR product was mixed with 4 ml of ExoSap-it (USB, Cleveland, OH, USA) and incubated at 37 C for 15 min followed by a denaturation step at 80 C for 15 min. Sanger sequencing was then performed with the BigDye V3.1 Terminator Kit (Applied Biosystems, Inc., Foster City, CA, USA). Single reactions contained 4 ml of Ready Reaction Mix, 0.2-mM primer, 2 ml 5Â Sequencing Buffer, 11.5 ml of nuclease free water (Applied Biosystems, Inc.) and 2 ml of post-Exosap-it PCR product. The sequencing reaction used the following thermocycling profile: 1 cycle of 94 C for 1 min; 25 cycles of 94 C for 15 s, 38 C for 30 s and 60 C for 4 min. Two microliters of sequencing reaction product was combined with 18 ml of Hi-Di Tm Formamide (Applied Biosystems, Inc.) and run on the ABI 3130 (Applied Biosystems, Inc.). The results were analyzed using Sequencing Analysis v5.2 (Applied Biosystems, Inc.).