女贞RAPD-PCR实验条件的优化

Optimization of the Experimental Conditions for RAPD-PCR of Ligustrum lucidum Ait.

采用单因素梯度试验对影响女贞(Ligustrum lucidum Ait.)RAPD扩增的若干重要影响因素(包括Mg Cl2、d NTPs、随机引物、Taq DNA聚合酶浓度、模板DNA用量,以及退火温度与循环次数等)进行了优化。优化后的女贞RAPD-PCR反应体系为:每25μL体积中含10×反应buffer 2.5μL,2.5 mmol·L-1Mg Cl23.0μL,0.2 mmol·L-1d NTPs 0.5μL,2.0 U Taq酶0.4μL,0.4μmol·L-1引物1.0μL,DNA模板60 ng。PCR扩增程序:在94℃条件下预变性4 min,然后依次在94℃条件下变性30 s,38℃条件下退火45 s,72℃条件下延伸2 min;进行40个循环,最后在72℃条件下延伸10 min,并于16℃条件下保存。以该优化的RAPD反应体系对木犀科女贞属植物不同居群间的女贞种质材料的扩增,均能获得丰富而清晰的条带,且重现性好。

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 ℃ for10 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.