Establishment and orthogonal optimization of ISSR-PCR amplification system in Salvia miltiorrhiza
LI Rong, WANG Zhe-Zhi( College of Life Sciences, Shaanxi Normal University, Xi’an 710062, China )
In this paper,the orthogonal design was used to optimize the ISSR-PCR amplification system of Salvia miltiorrhiza in four factors (the concentration of dNTP,Primers,Mg2+ and Taq DNA polymerase) at three levels respectively. Then,based on the optimal ISSR-PCR amplification system,the concentration of template DNA and annealing temperature were proposed by gradient PCR. The results showed that: a suitable ISSR-PCR amplification system of S.miltiorrhiza was established. In a total volume of 20 μL ISSR-PCR amplification system,it contains 1×PCR buffer,200 μmol/L dNTP,1.0 μmol/L primer,1.5 mmol/L Mg2+,1 U Taq DNA polymerase and 20～60 ng template DNA. The optimal annealing temperature for primer UBC 835 is 51.7 ℃.