茄子RAPD分子标记体系的建立与优化

Establishment and Optimization of RAPD-PCR Reaction System in Eggplant

采用改良的CTAB法提取茄子基因组DNA,并通过单因素多水平梯度试验,比较筛选RAPD扩增体系的各影响因素,建立了茄子RAPD-PCR的最佳反应体系:20μL反应体系其中含25 mmol/L MgCl2 2.0μL1、0×PCR Buffer 2.0μL、10mmol/L dNTP 0.5μL5、U/μL Taq E 0.2μL0、.1μmol/L Primer 3μL1、0 ng/L模板DNA 3μL、灭菌双蒸水9.3μL。扩增程序为:94℃预变性5 min;94℃变性1 min,37℃退火1 min7,2℃延伸1.5 min4,5个循环;72℃延伸10 min后4℃保存。

The genomic DNA of eggplant（Solanum melongena） was extracted by the modified CTAB method.The factors affecting RAPD results were studied through single factor and multi level tests.An optimal RAPD-PCR reaction system（20 μL） for eggplant was established,and it contained 25 mmol/L MgCl2 2.0 μL,10×PCR Buffer 2.0 μL,10 mmol/L dNTP 0.5 μL,5 U/μL Taq E 0.2 μL,0.1 μmol/L Primer 3 μL,10 ng/L template DNA 3 μL and water 9.3 μL.The appropriate amplification procedure was as follows： pre-denaturing for 5 min at 94 ℃,denaturing for 1 min at 94 ℃,annealing for 1 min at 37 ℃,elongating for 1.5 min at 72 ℃,45 cycles;elongating for 10 min at 72 ℃ and preserving at 4 ℃.