DNA extraction of birch leaves by improved CTAB method and optimization of its ISSR system

摘要

The basic method of DNA extraction (CTAB) was improved as the multi-times STE-CTAB extraction method and used to extract the DNA of birch leaved in this experiment. Results showed that the improved method is suitable not only for genomic DNA extraction of birch but also for that of other plants. The purity of genomic DNA extracted by the multi-times STE-CTAB extraction method is higher than that by one time STE-CTAB method, and it does not need the process of RNase. The factors of influencing ISSR system were explored based on the genomic DNA of birch extracted by the two methods. The optimal conditions for ISSR system were determined as follows: Mg2+ concentration is 1.5–3.0 mmol·L-1, dNTP concentration 0.10–0.25 mmol·L-1, the quantity of Taq polymerase 0.5–2.0 U, template DNA 30–100 ng, and the concentration of primer is 0.2?0.4 μmmol·L-1, and the reaction program was as: initial denaturation for 5 min at 94oC, 30 cycles of denaturation for 30 s at 94oC, annealing for 30 s at 51 oC, extension for 30 s at 72oC, and a final 7 min extension at 72 oC.