结球甘蓝RAPD反应条件的优化

Optimization of RAPD Reaction System for Cabbage

以甘蓝(Brassicaoleraceavar.capitata)基因组DNA为模板,比较筛选RAPD扩增体系的各影响因素,优化了甘蓝的RAPD反应体系,调整了扩增程序。该体系反应总体积为20μL,其中25mmol/LMgCl22.0μL,10×PCRBuffer2.0μL,10mmol/LdNTP0.5μL,5U/μLTaqE0.5μL,20ng/μLPrimer1.5μL,10ng/μL模板DNA3μL,灭菌双蒸水10.5μL。适宜的扩增程序为:94℃预变性5min;94℃变性1min,37℃复性1min,72℃延伸1.5min,40个循环;72℃延伸10min后15℃保存。

The genomic DNA of cabbage was used as a template, and the factors of influencing the RAPD reaction system were compared and selected so as to adjust the amplification process and optimize the RAPD reaction system. The results showed that the optimal RAPD reaction system with total volume of 20μL for cabbage was 2.0μL of MgC12 （25mmol/L） ＋ 2.0μL of PCR Buffer （ 10 × ） ＋ 0.5μL of dNTP （ 10 mmol/L） ＋ 0.5μL of Taq E （5U/μL） ＋ 1.5μL of Primer （20ng/μL） ＋ 3μL of DNA （10ng/μL） ＋ 10.5μL of H2O, and the appropriate amplification procedure was as follows： preparatorily denaturing for 5 rain at 94℃, denaturing for 1 rain at 94℃, annealing for 1 rain at 37℃, elongating for 1.5 rain at 72℃, 40 cycles, and elongating for 10 rain at 72℃.