Orthogonal design
Optimization of SCoT-PCR reaction system in Dactylis glomerata by orthogonal design
The effects of 5 factors (template DNA, Mg2+, dNTPs, Taq DNA polymerase, and primer) on the polymerase chain reaction (PCR) were investigated to optimize the start codon targeted polymorphism (SCoT)-PCR system of Dactylis glomerata L., using an orthogonal design L16 (45). A suitable SCoT-PCR system for D. glomerata was established; the 20 μL reaction volume contained 3.0 mM Mg2+, 0.2 mM dNTPs, 1.0 U Taq DNA polymerase, 0.2 μM primer, 20 ng template DNA, and 2 μL 10X buffer.
Extraction of total DNA and optimization of the RAPD reaction system in Dioscorea opposita Thunb.
Dioscorea opposita Thunb. has been used as health food and herbal medicinal ingredients in traditional Chinese medicine. In this study, the total DNA of D. opposita Thunb. was extracted using an improved cetyltrimethylammonium bromide (CTAB) method, and the extracted DNA was further used for random amplified polymorphic DNA (RAPD) reaction system by design of the L16 (44) orthogonal diagram.
Optimization of factors affecting Agrobacterium-mediated transformation of Micro-Tom tomatoes
Micro-Tom is the smallest known variety of tomatoes. An orthogonal experimental design L16 (45) was used to optimize Agrobacterium-mediated transformation of cotyledon explants of Lycopersicon esculentum cv. Micro-Tom. Four parameters were investigated to determine their effect on transformation frequency: the concentration of bacterial suspension, time of dip in bacterial suspension, co-cultivation time, and concentration of carbenicillin.