青钱柳ISSR-PCR反应体系的优化

Optimization of the ISSR-PCR Reaction System of Cyclocarya paliurus

采用改进的SDS法提取青钱柳基因组DNA,测试青钱柳ISSR扩增的最适退火温度,并采用单因素试验,测试了模板DNA、Mg2+、dNTP、BSA、引物\Taq酶6个因素对青钱柳ISSR扩增的影响。结果表明:合适的退火温度为56.3℃;适宜的扩增体系为:10μlPCR反应体积中,1×Taq酶配套缓冲液浓度为(10 mmol/LTris.HCl,pH值9.0,浓度为50 mmol/LKCl,0.1%Triton X-100),12 ng模板DNA,浓度为1.5 mmol/LMgCl2,浓度为1.0 mmol/L4×dNTP,1mg/ml BSA,15 pmol引物,0.75 UTaq酶。

The genomic DNA of Cyclocarya pdiurus was extracted with the improved SDS method. Bsed on its DNA, the ISSR amplificatory reaction system was optimized. The fitting annealing temperature was confirmed and the effect of Mg^2＋ concentration, dNTP concentration, BSA dosage, primer dosage, Taq DNA polymerase dosage and DNA templates dosage on ISSR amplification were tested with the single factor method. The result was that, the fitting annealing temperature was 56.3℃ and the optimal reaction system of ISSR for Cyclocaryapdiurus was determined as follows： 1 ×Taq polymerase corresponding buffer （10 mmol/L Tris·HCl pH 9.0,50 mmol/L KC1,0.1% Triton X-100）, 12ng template DNA, 1.5mmol/L MgCl2, 1.0 mmol/L 4×dNTP, 1mg/ml BSA, 15 pmol primer,0.75U Taq DNA polymerase in total 10 μl reaction volume, Thus, the establishment of ISSR-PCR reaction system settled the genetic diversity foundation for Cyclocaryapaliurus.