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采用改良CTAB法提取了桦褐孔菌总DNA,确定ISSR最适25μL反应体系为模板DNA浓度15 ng.μL-1,dNTPs浓度150μmol.L-1,引物浓度25μmol.L-1,Taq DNA聚合酶浓度2.0 U,Mg2+浓度1.4 mmol.L-1,10×buffer 2.5μL,其余用ddH2O补足;确定ISSR扩增程序为:94℃预变性5 min,35个循环:94℃变性1 min、45℃~51℃退火1 min(退火温度因不同引物而定)、72℃延伸1 min,最后72℃延伸5 min,4℃保存。筛选出16条ISSR引物,并成功应用引物UBC842完成了21株桦褐孔菌的ISSR-PCR反应。
The total DNA of Inonotus obliquus was extracted by modified CTAB method. The optimum 25μL reaction system of ISSR was determined as template DNA concentration 15 ng.μL-1, dNTPs 150μmol.L-1, primer concentration 25μmol.L-1, Taq DNA polymerase Enzyme concentration of 2.0 U, Mg2 + concentration of 1.4 mmol.L-1, 10 × buffer 2.5 μL, the rest with ddH2O make up; determine ISSR amplification program: 94 ℃ denaturation 5 min, 35 cycles: 94 ℃ denaturation 1 min, 45 Annealing at ℃ ~ 51 ℃ for 1 min (annealing temperature depends on different primers), extension at 72 ℃ for 1 min, extension at 72 ℃ for 5 min and preservation at 4 ℃. Sixteen ISSR primers were screened and ISSR-PCR of 21 Inonotus obliquus was successfully performed using primer UBC842.