Plant materials Seven banana (Musa species) cultivars Amritsagar, Sabri, Champa, Anupom, BARI kola-1, Mehersagar and Kabri were used in this research. One gram of young and healthy leaves from each sample was harvested for DNA isolation.
Reagents and solutions An extraction buffer consisting of 3 % CTAB (w/v), NaCl (2 M), 100 mM Tris-HCl pH 8.0 and 20 mM EDTA pH 8.0 was prepared. After being autoclaved for 20 min, 1.5% (w/v) PVP, 2 % (v/v) 2-mercaptoethanol were added to the extraction buffer, immediately before use. In addition, Phenol: chloroform: isoamyl alcohol (25:24:1, v/v/v), chloroform: isoamylalcohol (24:1, v/v), Ribonuclease A (10 mg/ml), proteinase K (20 mg/ml), Ethanol (70 %, 100 %), Sodium acetate (3M) solution (pH 8.0), and TE buffer (Tris HCl, 10 Mm,pH 8.0 , 1mM EDTA; pH 8.0) and absolute isopropanol were the additional solutions required.
DNA isolation protocol a. Freshly harvested young and healthy leaves of Musa species (1g) were ground to fine powder in presence of liquid nitrogen using mortar and pestle. b. The tissue powder was quickly transferred into a clear autoclaved 50 ml centrifuge tube and then 10 ml of pre-warmed (65 °C) CTAB-buffer was added and mixed very well. c. After that 40 µl of proteinase K ( 20 mg/ml) was added and mixed again by inversion for 1 min. d. The mixture was incubated at 70 °C for 30 min with gentle mixing by inverting the tubes. e. The tube was centrifuged at 13,000 rpm for 5 minutes and the supernatant was transferred to a clean centrifuge tube. f. An equal volume of phenol: chloroform: isoamyl alcohol (25:24:1, v/v/v) was added, mixed by using gentle inversion and centrifuged at 10,000 rpm for 5 min. g.The supernatant was transferred to a clean centrifuge tube and an equal volume of chloroform: isoamyl alcohol (24:1) was added and mixed properly by inverting the tubes 20-25 times to form an emulsion and centrifuged at 13000 rpm for 10 minutes. h. Again the supernatant was carefully decanted and transferred to a new tube and was precipitated by adding one volume of pre-chilled (-20oC) iso-propanol, mixed by gentle inversion incubated at -20oC for a minimum of one hour. i. The samples were centrifuged at 13,000 rpm for 15 minutes. The supernatant was discarded and the obtained pellet was washed twice and thrice with 70 % ethanol. Decanted the supernatant and air dried DNA pellet at room temperature until the whitish pellet turned to transparent. Finally, the pellet was resuspended in 300 μl of TE Buffer or deionized water and stored at -20ºC .In case of DNA contamination with RNA , an additional step needs to be performed, by adding 6μl of RNAase (10 mg/ml) and incubating it for 1 hour at 37ºC . All the centrifugation steps were carried out at room temperature to avoid precipitation with CTAB, DNA degradation and to obtain good quality of DNA.
Concentration, purity and quantity of the extracted DNA The quality and quantity of the extracted DNA were measured using a UV spectrophotometer at 260 nm and 280 nm. The purity of DNA was determined by estimating the ratio of absorbance at 260 nm to that of 280 nm. In order to verify DNA integrity, samples of DNA were subjected to gel electrophoresis on 1 %. Agarose gel, stained with ethidium bromide (Sambrook et al.1989) and compared with lambda DNA marker (used to determine the concentration). The nucleic acid concentration was estimated following (Sambrook et al.1989).