米心水青冈基因组DNA提取及RAPD反应体系优化

Genomic DNA Extraction and Optimization of RAPD Analytic Conditions of Fagus engleriana

采用4种方法对米心水青冈基因组DNA进行提取,通过比较得出改良CTAB法提出的DNA纯度较高,能够达到扩增要求,因此采用此方法用于正式DNA的提取。适合米心水青冈的RAPD反应体系为:反应体积为25μL,模板DNA40ng,引物0.8μmol.L-1,Taq聚合酶1.25U,Mg2+浓度2.0mmol.L-1,dNTP浓度0.16mmol.L-1。适合米心水青冈RAPD扩增程序:94℃预变性3min,一个循环,94℃变性30s,37℃退火1min,72℃延伸2min,45个循环。

Total DNA in leaf tissue of Fagus engleriana was extracted by four methods. Improved CTAB method was proved as the best one. The DNA sample obtained by this method was fit for RAPD experi- ment. RAPD reaction system（25 μL） ：40ng DNA template, 0.8 μmol · L^-1 primer, Taq DNA polymerase 1.25 U, 2.0 mmol · L^-1 Mg2＋ , 0. 16 mmol · L each dNTP. PCR amplification program was denaturation at 94℃ in advance for 3 min, then followed by 45 cycles, each cycle： denaturation at 94℃ for 30 s,anneal at 37℃ for I min and extention at 72℃ for 2 min.