Polygonum multiflorum ISSR-PCR reaction system and the establishment and optimization of.docVIP

 PAGE \* MERGEFORMAT 15 Polygonum multiflorum ISSR-PCR reaction system and the establishment and optimization of Authors: Zhen-Hua Zhao, Yan Ping, Jiao Xu-wen, Zhao jin [Abstract] Objective To establish and optimize the ISSR-PCR reaction system. Methods The study of single factor experiment ISSR reaction system main components (template, Taq DNA polymerase, Mg2, primers, dNTPs), as well as the results of annealing temperature on the amplification effects. The results established for the analysis of fleece-flower root ISSR reaction system and amplification process, ie 25 μ l reaction system containing 1 * Taq enzyme matching buffer, 100 ng template, 1.5 U Taq DNA polymerase, 1.5 mmol / L Mg2, 0.6 μ mol / L primer, 0.2 mmol / L dNTPs. Pre-amplification program was 94 ℃ denaturation 5 min; then 94 ℃ for 45 s, (according to different primer annealing temperature) annealing 1 min, 72 ℃ extension 1.5 min, 35 cycles; 72 ℃ extension 7 min, 4 ℃ . Conclusion The establishment of this optimization system for future use of ISSR markers to fleece-flower root identification and analysis of genetic diversity provides a standardized procedure. [Keywords:] Polygonum multiflorum; simple sequence repeat interval amplified too much sex; response system; establishment and optimization of Abstract: ObjectiveTo establish and optimize ISSR-PCR reaction system for Polygonum multiflorum Thunb.MethodsSingle factor experiment method was used to present the effect of the main reaction system elements (Taq DNA polymerase, template DNA, Mg2, primer, dNTPs) and annealing temperature on ISSR-PCR. ResultsA reaction system and amplified procedure suitable for Polygonum multiflorum Thunb. were established, that was, 25 μ l reaction system containing 1 * PCR buffer, 100ng template DNA, 1.5 U Taq DNA polymerase, 1.5 mmol / L Mg2, 0.6 μ mol / L primer, 0.2 mmol / L dNTPs. The optimal amplified procedure was as follows: after a pre-denaturing of 5 min at 94 ℃ , 35 cycles were performed with d