Optimization of the Experimental Conditions for RAPD-PCR of Ligustrum lucidum Ait.
doi: 10.15886/j.cnki.rdswxb.2014.04.012
- Received Date: 2014-10-08
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Key words:
- Ligustrum lucidum Ait. /
- RAPD /
- impact factors /
- system optimization
Abstract: Ligustrum lucidum Ait. was used as the experimental material for RAPD-PCR. Some important factors,such as the concentrations of Mg Cl2,d NTPs,random primers,Taq polymerase and template DNA as well as the annealing temperature and the cycles of amplification,were optimized by single factor experiment in order to establish the optimal RAPD-PCR system and procedure. Stable RAPD-PCR reaction parameters for L. lucidum Ait. were determined and optimized. 2. 5 μL 10×PCR buffer; 3. 0 μL 25 mmol · L-1Mg Cl2; 0. 5μL d NTPs,0. 4 μL Taq polymerase,1. 0 μL primers,and 60 ng DNA template were contained in 25 μL reaction solution. The PCR amplification program was to pre-denature at 94 ℃ for 4 min,then denature at 94 ℃ for 30 s,anneal at 38 ℃ for 45 s,extend at 72 ℃ for 2 min,and conduct 40 cycles with the last extension at 72 ℃ for 10 min. The amplified products were stored at 16 ℃. With this optimized RAPD reaction system the DNA of L. lucidum Ait. was amplified to produce rich,clear and repeatable bands.
Citation: | ZHAO Feng, LIU Guomin, LI Juanling, ZHANG Qiwen, FU Wei, ZHANG Enhua. Optimization of the Experimental Conditions for RAPD-PCR of Ligustrum lucidum Ait.[J]. Journal of Tropical Biology, 2014, 5(4): 374-380. doi: 10.15886/j.cnki.rdswxb.2014.04.012 |