黑木耳菌丝体DNA提取及RAPD扩增条件的优化

Optimization of DNA estraction and RAPD optimize amplification system of Auricularia auricula

对黑木耳菌株基因组DNA进行RAPD-PCR反应体系及扩增程序优化.结果表明,采用CTAB法快速提取到较高质量黑木耳基因组总DNA用于RAPD-PCR扩增,确定黑木耳基因组DNA RAPD-PCR最适25μL反应体系为:模板DNA浓度15 ng/μL,Mg2+浓度2.0 mmol/L,Taq DNA聚合酶1 U,dNTP浓度150μmol/L,10碱基引物浓度10 pmol,10×缓冲液,其余用重蒸馏水补充.扩增程序为:预变性94℃5 min,变性94℃1 min,退火36℃1 min,延伸72℃1.5 min,共40个循环,72℃最终延伸7 min.

Optimize the reaction system and procedural of RAPD, The results indicated that use the CTAB improvement method to withdraw high quality Auricularia auricula genome team total DNA to RAPD fast increasing. Determined that the RAPD--PCR Auricularia auricula most suitable 25μL reacting system is the template DNA density for 15 ng/μL, Mg2＋density 2.0 mmol/L, Taq DNA 1U, dNTP density 150 μmol/L, 10 basic group directs the density for 10 pmol, the 10×buffer solution density 2.5 mmol/L, other supple- mented with the heavy distilled water. Increases the procedure is： pre-denatured 94℃ 5 min, denatured 94 ℃ 1 min, annealing 36 ℃ 1 min, extends 72 ℃ 1.5 min, altogether 40 circulations; 72 ℃ extends finally 7 min.