OPTIMASI ISOLASI DNA DAN AMPLIFIKASI SEKUEN GEN trnL-F INTERGENIC SPACER cpDNA MANGGA (Mangifera) RIAU

Abstract

Molecular-based analysis of the plant depends on the amount and purity of DNA samples as well as optimum condition of amplification reaction in PCR. DNA isolation and PCR amplification is a common procedure in initiating the molecular-based analysis. There are various problems in the isolation of DNA and PCR amplification which need to be addressed because it determines the success of molecular-based research. Especially in isolating leaf samples containing higher plants secondary metabolites that affect the purity of the DNA. Mango (Mangifera) is a group of dicotyledonous plants contains high secondary metabolites that may contaminate obtained DNA. Therefore we need a proper method of DNA isolation and PCR amplification of Mangifera trnL-F intergenic spacer cpDNA. This study aimed to determine the appropriate methods in obtaining high molecular weight DNA from leaves of Mangifera Riau and to determine the optimum conditions for the PCR amplification of trnL-F intergenic spacer cpDNA. A total of 13 Mangifera species was isolated for their DNA using CTAB method. Amplification of their trnL-F intergenic spacer cpDNA was conducted using modified PCR condition of PCR procedure recommended by the primer producer and taq Hot Start Green PCR Master Mix kit.Total DNA of all Mangifera showed various quantity and quality. The total quantity of DNA which obtained were range between 50-100 ng / ml. The quality of DNA molecules are thick, intact and without smear. PCR amplification method of gene sequences trnL-F intergenic spacer cpDNA from 13 species of Mangifera produced DNA sequence of 420 bp.