For long-range genomic PCR, total genomic DNAs were harvested using DNeasy Blood & Tissue kit (Qiagen; catalog number: 69504), and long-range PCR reactions were performed using Q5 High-Fidelity 2× Master Mix (New England Biolabs; catalog number: M0492). We used 100 ng genomic DNAs as the template, and the PCR conditions were first 1 cycle of 98 °C for 30 s, followed by 40 cycles of 98 °C for 10 s, 66 °C for 30 s, and 72 °C for 2 min. The PCR products were subjected to 1% agarose gel electrophoresis and the DNA bands of interest were purified using QIAquick Gel Extraction Kit (Qiagen; catalog number: 28706) and subjected to direct Sanger sequencing (Genewiz) and analyzed using FinchTV (Geospiza).
For RT-PCR, total RNAs were harvested using RNeasy Mini kit (Qiagen; catalog number: 74106) and cDNAs were made using QuantiTect Reverse Transcription kit (500 ng RNA; Qiagen; catalog number: 205311). The cDNAs were then subjected to PCR reactions using Q5 High-Fidelity 2× Master Mix and the PCR conditions were: first 1 cycle of 98 °C for 30 s, followed by 40 cycles of 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min. The PCR products were subjected to 1% agarose gel electrophoresis and the DNA bands of interest were purified using QIAquick Gel Extraction Kit and subjected to direct Sanger sequencing (Genewiz) and analyzed using FinchTV (Geospiza).
For RT-PCR, total RNAs were harvested using RNeasy Mini kit (Qiagen; catalog number: 74106) and cDNAs were made using QuantiTect Reverse Transcription kit (500 ng RNA; Qiagen; catalog number: 205311). The cDNAs were then subjected to PCR reactions using Q5 High-Fidelity 2× Master Mix and the PCR conditions were: first 1 cycle of 98 °C for 30 s, followed by 40 cycles of 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min. The PCR products were subjected to 1% agarose gel electrophoresis and the DNA bands of interest were purified using QIAquick Gel Extraction Kit and subjected to direct Sanger sequencing (Genewiz) and analyzed using FinchTV (Geospiza).