Optimization of SCoT-PCR reaction system in Dactylis glomerata by orthogonal design.
نویسندگان
چکیده
The effects of 5 factors (template DNA, Mg(2+), dNTPs, Taq DNA polymerase, and primer) on the polymerase chain reaction (PCR) were investigated to optimize the start codon targeted polymor-phism (SCoT)-PCR system of Dactylis glomerata L., using an orthogo-nal design L16 (4(5)). A suitable SCoT-PCR system for D. glomerata was established; the 20 μL reaction volume contained 3.0 mM Mg(2+), 0.2 mM dNTPs, 1.0 U Taq DNA polymerase, 0.2 μM primer, 20 ng tem-plate DNA, and 2 μL 10X buffer. Each factor had a different effect on the amplification reaction, and the concentration of dNTPs had the larg-est effect on the SCoT-PCR system. We tested 10 orchardgrass samples to determine and verify the stability of the reaction system. The results showed that amplified bands from diverse materials were clear, stable, and rich in polymorphisms, indicating that the optimized system was very stable.
منابع مشابه
Orthogonal design in the optimization of a start codon targeted (SCoT) PCR system in Roegneria kamoji Ohwi.
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ورودعنوان ژورنال:
- Genetics and molecular research : GMR
دوره 14 2 شماره
صفحات -
تاریخ انتشار 2015