漾濞核桃RAPD反应优化体系的建立

Establishment of Optimal Reaction System of RAPD for Juglans Silillata

以漾濞核桃中的大姚三台核桃为实验材料,通过对影响RAPD扩增结果的主要因子Tag酶、Mg+2、引物、模板DNA、dNTPs等不同的浓度和组合的试验研究,确定了漾濞核桃的最适反应体系和扩增程序,即在25μL反应体系中,0.75 u.L-1Taq DNA聚合酶,3.0 mmol.L-1Mg+2,0.3 mmol.L-1引物,含1.6 mg.L-1模板,2.5 ul 10×Buffer,dNTPs各0.2 mmol.L-1。扩增程序为:94℃预变性300 s,94℃变性40 s,36℃退火60 s,72℃链延伸120 s,45次循环后,72℃延伸600 s。

Establishment of optimal reaction system of RAPD is the basic work for identifying the different walnut varieries by the ways of molecular markers. The leaves of Juglans silillata as material, factors of：Taq DNA polymerase, Mg^＋ 2, tenmplate D NA, primers and dNTPs which will influence the results of RAPD. were studied in the experiment. Moreover an optimal reaction system and the PCR reaction program have been established. Reaction system is 25 ul amplification reaction solution consisting of 0.75 u·L^-1 Taq DNA polymerase, 0.3 mmol·L^-1 Mg^＋2, 0.3 mmol·L^-1 primer, 1.6 mg·L^-1 tenmplate DNA, 2.5 ul 10 ~ Buffer and 0.2 mmol＂ L-1 dNTPs. The PCR amplification program is predenaturing at 94℃for 300 s, followed by denaturing at 94℃ 60 s, anneling 36℃ for 60 s, extension 72℃ for 120 s, cycling 45 times, last extension 72℃ for 600 s.