Qingtiankui total DNA extraction and random amplified polymorphic DNA reaction conditions the establishment of.doc

 PAGE \* MERGEFORMAT 12 Qingtiankui total DNA extraction and random amplified polymorphic DNA reaction conditions the establishment of [Abstract] Objective To establish and optimize Qingtiankui the total DNA sample extraction and random amplified polymorphic DNA (RAPD) reactions. Methods to improve the high-salt, low pH extraction Qingtiankui samples of fresh leaves and herbs, through the orthogonal design and uniform design, changes in procedures for optimization of PCR reaction system and the random amplified polymorphic DNA. The results using the high-salt low-pH value method may be better fresh leaf extract DNA, medicinal leaves contain a lot of impurities, but can be used for RAPD. Reaction system as follows: buffer 2.0 μ 1, 0.5 μ l dNTP (each 2.5 mmol * L-1), 0.8 μ l Mg2 (25 mmol * L-1), 0.5 μ l primers (20 pmol * μ l-1), 0.2 μ l Taq enzyme (5U * μ 1-1), 1 μ l DNA (50 ng), 1μ l BSA (20 mg * ml-1), plus double-distilled water to 20 μ l. Amplification procedure: 94 ℃ pre-denaturation 3 min; 94 ℃ denaturation 30 s, 40 ℃ annealing 30 s, 72 ℃ extension 60 s, 40 cycles; 72 ℃ extension of 5 min. Conclusion established and optimized Qingtiankui sample DNA extraction method and the total RAPD reaction system. [Keywords:] Qingtiankui random amplified polymorphic DNA extracted DNA orthogonal design uniform design Abstract: ObjectiveTo establish and optimize the methods of total DNA extraction and RAPD analysis of Nervilia fordii. MethodsLow pH extraction medium with high salt method was adopted to extract genomic DNA from fresh and medicinal material leaves of Nervilia fordii, and the randomly amplified polymorphic DNAs ( RAPD) technique was optimized through changing PCR reaction system. ResultsLow pH extraction medium with high salt was better to extract fresh leaves than medicinal material leaves, but both of them could be suitable to RAPD. The optimal RAPD conditions was as follows: 20μ l solution contains 1 * Buffer solution, 0.5μ l dNTP (each2.5mM