蓖麻标雌/单雌/两性系花序MSAP反应体系的建立

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。

Establish Castor Marked Female Lm Per Female Amphoteric Lineinflores Cence MSAP Reaction System

The purpose of this experiment was to establish Lm-type female system castor different developmen-tal stages marked female,per female,amphoteric system inforescence establish MSAP reaction system.MSAP reac-tion system established to determine differences in inflorescence type genes at different developmental stages of re-search to lay the foundation.The results were as follows:extraction of different types at different developmental sta-ges of inflorescence genomic DNA mixed into the gene pool.EcoRⅠ and HpaⅡ /MspⅠ digested gene pool 12 h. The digestion products and EcoRⅠ,Hpa Ⅱ /MspⅠ adapter overnight connection for pre-amplification.The opti-mized system for the pre-amplification reaction 10 ×PCR Buffer 2.5 μL,dNTPs (10 mmol /L)2.5 μL,Mg2 +(25 mmol /L)2.0 μL,template 2.0 μL,E 0 amplification primer (10 pmol /μL)1 .5 μL,H0 amplification primer (10 pmol /μL)1 .5 μL,LA Taq(5 U /μL)0.25 μL,ddH2 O 12.75 μL.Pre-amplification products were diluted 10-fold for the selective amplification.Selective amplification system optimized for 10 ×PCR Buffer 2.5 μL,dNTPs (10 mmol /L)2.0 μL,Mg2 + (25 mmol /L)4.0 μL,template 3.0 μL,E X amplification primer (10 pmol /μL ) 1 .0 μL,H /Mx amplification primer(10 pmol /μL)1 .0 μL,LA Taq(5 U /μL)0.30 μL,ddH2 O 1 1 .2 μL.