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为了建立Lm型雌性系蓖麻不同发育时期的标雌/单雌/两性系花序MSAP反应体系,提取不同类型不同发育时期的花序基因组DNA,混合成基因池;用EcoRⅠ和HpaⅡ/MspⅠ的组合,12 h可将DNA酶切完全;16℃条件下,将酶切产物与EcoRⅠ、HpaⅡ/MspⅠ接头过夜连接;连接产物用于预扩增。优化后的预扩增反应体系为10×PCR Buffer 2.5μL、d NTPs(10 mmol/L)2.5μL、Mg2+(25 mmol/L)2.0μL、模板2.0μL、E0扩增引物(10pmol/μL)1.5μL、H0扩增引物(10 pmol/μL)1.5μL、LA Taq(5 U/μL)0.25μL、dd H2O 12.75μL。预扩增产物稀释10倍后用于选择性扩增。优化后的选择性扩增体系为10×PCR Buffer 2.5μL、d NTPs(10 mmol/L)2.0μL、Mg2+(25mmol/L)4.0μL、模板3.0μL、EX扩增引物(10 pmol/μL)1.0μL、H/MX扩增引物(10 pmol/μL)1.0μL、LA Taq(5U/μL)0.30μL、dd H2O 11.2μL。
In order to establish a MSAP reaction system of female / single female / amphiploid inflorescence at different developmental stages of Lm female castor, different genomic DNAs of inflorescences of different developmental stages were extracted and mixed into gene pool. The combination of EcoRⅠ and HpaⅡ / MspⅠ, 12 h DNA digestion can be complete; under 16 ℃ conditions, the enzyme cut EcoRI, Hpa Ⅱ / Msp Ⅰ linker overnight connection; ligation products for pre-amplification. The optimized pre-amplification reaction system was 2.5 μL of 10 × PCR Buffer, 2.5 μL of dNTPs (10 mmol / L), 2.0 μL of Mg2 + (25 mmol / L), 2.0 μL of template and 10 pmol / μL of E0 amplification primer 1.5 μL, 1.5 μL of H0 amplification primer (10 pmol / μL), 0.25 μL of LA Taq (5 U / μL) and 12.75 μL of ddH2O. Preamplification products were diluted 10-fold for selective amplification. The optimized selective amplification system was 2.5 μL of 10 × PCR Buffer, 2.0 μL of dNTPs (10 mmol / L), 4.0 μL of Mg2 + (25 mmol / L), 3.0 μL of template and 10 pmol / μL of EX amplification primer 1.0 μL, 1.0 μL of H / MX amplification primer (10 pmol / μL), 0.30 μL of LA Taq (5 U / μL) and 11.2 μL of ddH2O.