Optimization of annealing temperature for amplification of EhoscnOla locus in pranajiwa (Euchresta horsfieldii) plant collected from mountains, urban and coastal areas in Bali

Abstract

Pranajiwa plant is a medicinal plant that grows wildly and is classified as a rare plant. Currently, its existence is increasingly threatened. Pranajiwa grows around Indonesia and is known with several scientific names and morphological features due to unclear identification. Molecular identification is recommended to clarify its species. DNA Barcoding is considered the suitable method to identify pranajiwa plant molecularly. The purpose of this study was to optimized the PCR annealing temperature of EhcSnOla locus barcoding marker of pranajiwa plants collected from the coastal (Jimbaran), urban (Renon), and mountain (Bedugul) areas, representing three different areas in Bali. Research procedures include total DNA extraction, PCR procedure, and electrophoresis. The primers used in this study were EhoScnOla forward primer and EhoscnOla reverse primer. Five different temperatures were used for annealing temperature optimization: 51 C, 52 C, 55 C, 57 C, and 60 C. The result showed that all temperatures produced a clear, thick, and single electrophoresis band, indicating that all temperatures were suitable for the annealing temperature and the most optimal temperature is in the Mountains sample (Bedugul) which is 60 C. The Jimbaran, Renon, and Bedugul samples produced 882, 820, and 889 bp, respectively. EhcSnOla locus can be used as the barcoding marker to identify pranajiwa molecularly.