Isolation of DNA suitable for the random amplification of polymorphic DNA of mango.
Saldaña G. A., Salazar E. G.
Author Affiliation: INIA, Centro Nacional de Investigadores Agropecuarias (CENIAP), Edif. 09. Campo Experimental CENIAP, Unidad de Biotecnolog´a Vegetal, Zona Universitaria, via El Limón, Maracay 2101, estado Aragua, Venezuela.
Agronomía Tropical (Maracay) 57 : 281-286
Abstract : Leaf tissue DNA of 49 accessions of the Mango Germplasm Collection of the National Centre for Agricultural Research was characterized using random amplified polymorphic DNA. Calibration of buffer extractions was undertaken by comparing 4 lysis solutions and initial conditions for incubating the macerated tissue. DNA precipitation conditions, DNA pellet washing solutions and effect of lyophilization on DNA pellet resuspension were also studied. Of the 4 DNA extraction protocols, the highest amounts of DNA were obtained with the Doyle and Doyle (1990) methodology. The best results were obtained by macerating 250 mg of leaf tissue with 900 µl 2X CTAB-TRIS base lysis buffer, preheated at 60°C. All the solutions were incubated at 60°C for one hour. Storing the macerate at 20°C for 24 h increased the amount of DNA precipitation when cold 98% isopropanol was used. Washing pellets at 37°C with 70% ethanol produced white sediments. Finally, improvements in the resuspension of the DNA pellet in TE buffer occurred when tissues were lyophilized at 50°C for 3 h. The methodology proved effective at isolating DNA from all the studied cultivars. The DNA concentration was higher than 350 ng/µl in all cases.