腐食酪螨ISSR最佳反应体系的设计

本文以腐食酪螨基因组DNA为模板,采用逐个参数优化法,探讨腐食酪螨ISSR实验的最佳反应体系。实验结果表明,腐食酪螨ISSR-PCR最佳反应体系:总体积25μL,其中Mg2+1.5 mM;10×Buffer 2.5 mM;dNTPs 0.2 mM;ISSR引物0.2μM;模板DNA 50 ng;Taq DNA聚合酶0.75 U。扩增程序为:94℃预变性5min,94℃变性1 min,(48℃、50℃、52℃)复性1 min,72℃延伸1.5 min,循环35次,结束后72℃延伸5 min,4℃保存。同时通过梯度退火实验,确定不同引物的最佳退火温度。

Optimization for ISSR Reaction System of Tyrophagus putrescentiae(Schrank)

Based on the genomic DNA of Tyrophagus putrescentiae（Schrank）,the factors influencing ISSR system were explored one by one.The results showed that the optimal conditions for ISSR reaction system（25μl） were determined as the following： 1.5mmol/L of Mg2＋,0.2 μmol/L of dNTPs mixture,0.2 μmol /L of each primers,50 ng of template DNA,1.0 U of Taq DNA polyerase.The reaction program was as： initial denaturation for 5min at 94℃,35 cycles o f denaturation for 1min at 94℃,annealing for 1min at 48℃,50℃ and 52℃,extension for 1.5m in at 72℃,with a final extension of 5min at 72℃.The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR.Total of 40 primers subjected to three different annealing temperatures.