Chapter 3.1 Total Rna Extraction From Cacao
Chapter 3: Methods 3.1 Total RNA extraction from cacao tissue. 3.1.1 RNA extraction and purification The plant leaves are collected from Taman Koko, Tawau, Sabah. The RNA extraction of T.cacao are performed using tertiary-butanol protocol. Mortars, pestles and spatula were soaked overnight in a 0.1% (v/v) solution of diethylpyrocarbonate (DEPC) and then were autoclaved before use. Eighty milligrams of cacao leaves were grind in liquid nitrogen and then transferred into a 2µL sterile microcentrifuge tube. Next, 1 mililitre of homogenization buffer (0.2 M boric acid, 10 mM EDTA, pH adjusted to 7.6 with Tris), 20 µL of 25% of sodium dodecyls sulfate (SDS), and 20 µL of 14.3 M B-mercaptoethanol were added. The mixture was shaken for 10 min and then centrifuged at 15,000× g at room temperature for 10 min. …show more content…
Finally, the supernatant was extracted with 1 mL chloroform/isoamyl alcohol (24:1; v/v). The separation of the nucleic acids from the polysaccharides was made by the addition of 70 µL 3 M sodium acetate, pH 4.5, and 308 µL tertiary (tert)- butanol (0.4 volumes) to the upper aqueous phase (700
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