Leucocephala Case Study

Malaysia is one of the countries with the most abundant types of tropical tree species available in nature. Woods and timber are important in the economic development of Malaysia especially in the import and export industry. One type of woods with such vital function is Leucaena leucocephala. It is a plant species with many branches and numerous clusters of flat pods that enveloped the seeds (Shelton et al., 1994). L. leucocephala was first brought into Southeast Asia in the last few centuries by the Spanish for the purpose of livestock feeding. In Malaysia, it is commonly known as “petai belalang”. Aside from being used for furniture production, paper making, timber in construction and charcoal, L. leucocephala is also beneficial as a shade
Approximately about 1 g of leaf tissue was frozen with liquid nitrogen and ground into fine powder using mortar and pestle. Then, the plant powder was transferred into a 15 mL Falcon tube that contained the preheated CTAB extraction buffer. The sample was then incubated for 2 hours at 60°C. After incubation, the sample was allowed to cool to ambient before adding an equal volume of CIA (24:1). It was mixed gently for 15 minutes and centrifuged at 4, 000 rpm for 15 minutes to obtain supernatant. After centrifugation, the supernatant was transferred to a new 15 mL Falcon tube and an equal volume of CIA was added. The mixture was mixed gently and centrifuged again for 15 minutes at 4, 000 rpm. Next, the aqueous phase (top layer) was transferred to a clean 15 mL Falcon tube and 2/3 volume of cold isopropanol (-20°C) was added. The sample was stored overnight at
Therefore, purification using PCIA was performed as the mixture of phenol and chloroform can remove proteins, lipids, carbohydrates, and cell debris from the DNA (Tan & Yiap, 2009). Repetitive CIA treatment was also done to ensure the elimination of chlorophyll, pigments, and dyes from the genomic DNA (Sahu et al., 2012).
Ethanol or isopropanol was added to the supernatant to allow the precipitation of DNA (Choudary et al., 2008; Tan & Yiap, 2009). Centrifugation was done several times to remove insoluble particles and also to purify the DNA. In order to remove excess salt, DNA was rinsed with 70% ethanol (Tan & Yiap, 2009). RNAse was also added to decrease the amount of RNA present in the genomic DNA (Choudary et al., 2008). Having a high concentration of RNA in the sample may lead to chelation of magnesium ion that eventually causing reduction in PCR yield (Padmalatha & Prasad,