Abstract:
Salacia spp. belong to the family Celastraceae, with some species native to Sri
Lanka and listed as endangered or near threatened on the National Red List 2012 of
Sri Lanka. The higher content of secondary metabolites in this plant hindered the
extraction of sufficient quantities of good-quality DNA for downstream processes
like PCR and DNA sequencing. Therefore, this research aimed to optimize the DNA
extraction protocol to obtain a high amount of high-quality DNA from Salacia sp. in
the absence of liquid Nitrogen. DNA was extracted using sixteen different methods
fewer than two main protocols based on the CTAB miniprep procedure. Three
replicates were maintained for each method. The quality and quantity of extracted
DNA were measured using a nano spectrophotometer and verified by
electrophoresis. The best protocol for obtaining high-quality DNA from Salacia sp.
involves the following steps: Immature, healthy leaf samples were collected and
stored at -80°C. The frozen samples were ground using a pre-cooled mortar and
pestle. The ground material was then mixed with a preheated (60°C) CTAB buffer
containing PVP and incubated for 30 minutes. After incubation, the mixture was
centrifuged at 13,000 rpm for 10 minutes, and the supernatant was treated with 500
μL of chloroform, followed by another centrifugation at 13,000 rpm for 10 minutes.
This extraction step was repeated. The resulting supernatant was mixed with
absolute ethanol and ammonium acetate and stored at -20°C overnight. The next
day, the mixture was centrifuged at 13,000 rpm for 15 minutes. The pellet was
washed with 70% ethanol, dried, and dissolved in ultra-pure water. This protocol
effectively yielded the highest DNA concentration (0.636±0.339μg/μL), with an
A260/A280 ratio of 2.008 ±0.041, indicating minimal contamination among the
sixteen different samples. A distinct DNA band was observed on a 0.8% agarose gel
using that method, confirming the quality of DNA. Therefore, this modified method
successfully enabled the isolation of good-quality DNA from Salacia sp. with a
considerable yield.
