山茶ISSR扩增条件的优化

Optimization of the ISSR reaction system of Camellia japonica

以山茶基因组DNA为材料,测试山茶ISSR扩增的最适退火温度,并采用单因素试验,测试了模板DNA量,Mg2+浓度,dNTP浓度,BSA浓度,引物用量,Taq酶用量等6个因素对山茶ISSR扩增的影响。结果显示:山茶ISSR扩增的最适退火温度为56.3℃;适宜的扩增体系为:10μL PCR反应体积中,1×Taq酶配套缓冲液(10 mmol.L-1Tris.HCl pH 9.0,50mmol.L-1KCl,0.1%Triton X-100),2 mmol.L-1MgCl2,0.6 mmol.L-14×dNTP,2 mg.ml-1BSA,16 ng模板DNA,10 pmol引物,0.5 U Taq酶。

The suitable ISSR-PCR amplification condition of the genomic DNA of Camellia japonica was optimized and the optimal annealing temperature was determined. Using single factor test method, the effects of 6 factors, such as Mg^2＋ concentration, dNTP concentration, BSA dosage, DNA templates dosage, primer dosage and Taq DNA polymerase dosage for ISSR amplification were tested, The optimal annealing temperature was 56.3℃ and the optimal ISSR reaction system for Camellia japonica was as follows： 1 × Taq polymerase buffer [10 mmol·L^-1Tris.HCl pH 9.0,50mmol·L^-1KCl,0.1%Triton X-100], 2 mmol·L^-1 MgCl2, 0.6 mmol·L^-1 4 6 dNTP, 2 mg·ml^-1 BSA, 16 ng DNA template, 10 pmol primer, 0.5 U Taq DNA polymerase in total 10μL reaction volume.