摘要
分别以砀山酥梨不同品系的成熟叶片、幼叶和芽为材料,采用SDS法提取基因组DNA,比较了不同材料的提取效果,并以此为模板进行RAPD反应,优化了RAPD反应条件。结果表明:用幼叶和芽均能提取高质量的基因组DNA,可直接用于RAPD分析,而用成熟叶片虽能提取到基因组DNA,但不能直接用于RAPD分析;优化的RAPD反应体系为:反应体积50μL,包含80ng左右模板DNA、5μL市售10×PCRBuffer、2.0mmol/LMgCl2、120μmol/LdNTPs、3U的Taq酶、0.5μmol/L随机引物;热循环程序为94℃预变性5min,使模板DNA充分变性,然后进入下列温度循环,94℃变性30s,36℃退火45s,72℃延伸90s,循环数40,最后72℃延伸7min。
The isolation effect of genomic DNA from mature leaf, tender leaf and bud of Dangshansu pear was studied respectively by SDS method. After comparing the quantity and quality of genomic DNA isolated from different materials,the highquality DNA was used as template to optimize the reaction condition of RAPD analysis system. The result showed that the genomic DNA isolated from tender leaf and bud could be used for RAPD analysis and that isolated from mature leaf couldn't be used directly. The optimum reaction condition of RAPD analysis system for Dangshansu pear was as follows: total reaction volume 50μl, including about 80 ng template DNA,5 μL 10×PCR Buffer purchased, 20 mmol/L MgCl2,120 μmol/L dNTPs,3 unite Taq polymerase, 05 μmol/L random primer; the program of heating:predenaturation 5 min at 94℃ to make template DNA denaturating sufficiently,then undergo a proceeding of reaction cycles as denaturation 30 s at 94℃,anneal 45 s at 36℃, polymerization 90 s at 72℃, 40 cycles, finally polymerization 7 min at 72℃。
出处
《安徽农业大学学报》
CAS
CSCD
北大核心
2003年第2期178-181,共4页
Journal of Anhui Agricultural University
