小麦基因组SRAP扩增体系的初步研究

对影响SRAP扩增结果的各种因素包括DNA、引物、dNTPMixture、TaqDNA聚合酶的浓度及变性、复性、延伸的时间及温度进行了摸索。获得了能够稳定扩增小麦基因组的体系:20μL体系中DNA25ng,dNTP188mmol/L,TaqDNA聚合酶0 75U,引物25ng。扩增程序为:94℃1min,37℃1min,72℃10sec,5个循环;94℃1min,50℃1min,72℃10sec,35个循环;72℃1min。

Establishment of SRAP technique system in wheat genome

In this paper, some factors involving genomic DNA template, dNTP Mixture, Taq polymerase and PCR amplification time and temperatures of denature, annealing and amplification affecting the SRAP researching system of wheat were studied and an advanced technical system was established. The 20 μL reaction mixture contained: 25 ng of genomic DNA template, 188 mmol/L of dNTP, 0.75 U of Taq polymerase, 25 ng of primer and appropriate reaction buffer. Amplification was carried out in two steps: in the first 5 cycles, the annealing temperature was 37℃, and during the following 35 cycles, the annealing temperature was 50℃. In all the cycles, denaturation was carried out for 1 min at 94℃, annealing for 1 min, and amplification for 10 sec at 72℃. After all the cycles, amplification was done for 1 min at 72℃.