Dnaeasy mini kit

Genomic DNA was isolated from the EDTA blood by using a Qiagen DNAeasy mini kit. The isolated DNA was then amplified by PCR using TPO gene specific primers that together covered from Exon 8 to Exon 14, since global data showed that most of the common mutations in the TPO gene of the patients with congenital hypothyroidism were confined in this region. The primer sequences are listed in Table 1.
To amplify the desired target sequence of TPO gene, PCR amplification was conducted on a thermal cycler (Bio-Rad, USA). The final reaction volume was 10 µl for each of the reactions which contained 1 µL 10X PCR buffer, 0.3 µL 25 mM MgCl₂, 2 µL 5X Q-solution, 1.6 µL 2.5 mM dNTPs mixture, 0.2 µL 10 mM Forward and 0.2 µL Reverse primers, 0.05 µL Taq DNA Polymerase, and 50 ng of genomic DNA, and total reaction volume was made up to 10 µL by addition of nuclease free water. The thermal cycling condition included (a) initial denaturation at 95°C for 5 minutes, (b) 35 cycles of denaturation at 95°C for 40 seconds annealing at 58°C for 35 seconds and extension at 72°C for 40 seconds, and (c) final extension at 72°C for 5 minutes.

Putative transformants and the wild-type strain of the C. macilenta mycobiont were grown in 30 mL of MEB medium at 15 °C in an orbital shaker (200 rpm) for 7 days for genomic DNA extraction. The mycobiont was harvested and ground into a fine powder under liquid nitrogen. DNA was extracted using DNAeasy mini kit, according to the manufacturer’s instruction (Qiagen, Valencia, CA, USA).