Abstract:

To establish a stable, repeatable ISSR-PCR reaction system of Mytilaria laosensis, those five factors, the concentration of DNA template, dNTPs, Mg2+, and primers and Taq DNA polymerase, were studied through the single factor tests, orthogonal tests and variance analysis with the total DNA of Mytilaria laosensis as experimental material. The optimal system of ISSR-PCR reaction was established as follows: 30 ng template DNA, 0.20 mmol/L dNTPs, 2.75 mmol/L Mg2+, 0.6 μmol/L primers, 2.20 U Taq DNA polymerase were contained in 20μL reaction system. Variance analysis showed that: only the Mg2+ concentration of five factors has the greatest impact on the reaction system and reached a significant level.