Dna extraction, , PCR

Ghosh, Arnab, Bharti, Daizy, Bhutiani, Rakesh, Pathania, Prakash Chand & Kumar, Santosh, 2025, Morphology, morphogenesis, and molecular phylogeny of an Indian population of Notohymena quadrinucleata Foissner, 2016 (Ciliophora: Oxytrichidae), Journal of Natural History 59 (25 - 28), pp. 1917-1931 : 1919

DNA extraction, PCR amplification and sequencing

About 100 cells were collected from an overnight starved clonal culture with the help of glass micropipettes and washed three times with autoclaved distilled water. Genomic DNA was extracted (50 µL) using the DNeasy blood and tissue kit (Qiagen, GmbH, Hilden, Germany), following the manufacturer’s instructions. Extracted DNA was amplified using the universal eukaryotic primers Euk A (FW 5’- AACCTGGTTGATCCTGCCAGT-3’) and Euk B (RV 5’-TGATCCTTCTGCAGGTTCACCTAC- 3’) ( Medlin et al. 1988). The Polymerase Chain Reaction (PCR) program for 18S rDNA amplification included an initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30s, 50°C for 30s and 72°C for 80s, with the final extension of 7 min at 72°C. After confirmation of the appropriate size, the PCR products were purified using the QIAGEN QIAquick PCR Purification Kit and directly sequenced on both strands using Applied Biosystems 3730xL analyser at Wipro Limited, Bengaluru, India. Contigs were prepared by manual editing.