3500 dna analyzer

To study the patterns of genetic diversity and genomic admixture in sympatric and allopatric populations, we used 15 nuclear microsatellite loci (SSR) previously developed for other bromeliad species. Six loci were isolated from V. simplex (Vs1, Vs2, Vs8, Vs9, Vs10 and Vs19; Neri et al. 2015 ), six loci from V. gigantea (VgB01, VgB10, VgB12, VgG02, VgG04 and VgG05; Palma-Silva et al. 2007 ), two loci from Tillandsia fasciculata (E6 and E6b; Boneh et al. 2003 ) and one locus from Aechmea caudata (Ac01; Goetze et al. 2013 ). For each SSR, the forward primers were synthesized with an M13 tail (5′-CACGACGTTGTAAAACGAC-3′) to allow for marking and multiplexing fluorescent dyes during the amplification and genotyping procedures. All PCR amplification reactions were performed in a Thermal Cycler (Applied Biosystems, Foster City, CA, USA) following the protocol described by Palma-Silva et al. (2007) . The microsatellite alleles were resolved on an 3500 DNA Analyzer automated sequencer (Applied Biosystems) and sized against the GeneScan 500 LIZ molecular size standard (Applied Biosystems) using GENEMARKER Demo version 1.97 software (SoftGenetics, State College, PA, USA). Microchecker software (Van Oosterhout et al. 2004 ) was used to check for null alleles.

The hotspot regions of the oncogenes EGFR (exons 18, 19, 20 and 21) and KRAS (codons 12/13) were analyzed by polymerase chain reaction (PCR), followed by direct sequencing, as previously described [17 (link)]. Briefly, PCR was performed in a final volume of 15 μl, with 50 ng of DNA and 10 μM of forward and reverse primers, using 7.5 μl of the HotStar master mix (Qiagen, Hilden, Germany) according to the protocol proposed by the manufacturer, with the following cycling parameters: 96 °C for 15 min, followed by 40 cycles of 96 °C for 45 s, 58 °C for 45 s (EGFR) or 56.5 °C for 45 s (KRAS), 72 °C for 45 s and 72 °C for 10 min in a thermocycler (Veriti, Applied Biosystems, Carlsbad, USA). Primer sequences were previously described [15 (link)]. The PCR products were evaluated by electrophoresis in agarose gel and further purified using ExoSAP-it (Affymetrix), followed by cycle sequencing carried out using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) with an initial denaturation at 97 °C for 3 min, followed by 28 cycles of 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. Sequencing products were purified using BigDye Xterminator (Applied Biosystems) and analyzed on a 3500 DNA Analyzer with a ABI capillary electrophoresis system (Applied Biosystems). Sequences were analyzed using the SeqScape software package (Applied Biosystems).