RNA酶保護試驗((RNaseProtectionAssay,RPA)方法
一、試劑準備1． GACU POOL：取100mM ATP、CTP、GTP各2.78μl、100mM UTP 0.06μl，加DEPC H2O至100μl。2. 雜交緩沖液IPES 0.134g、0.5M EDTA（pH8.0）20μl、5M NaCl 0.8ml、甲酰胺8ml，加DEPC H2O至10ml。3. RNase消化液：5M NaCl 120μl、1M Tris-HCl(pH7.4) 20μl、0.5M EDTA（pH8.0）20μl、RNase A(10mg/ml) 8μl、RNase T1(250U/μl) 1μl，加DEPC H2O至2ml二、操作步驟1．反義RNA可由含T7或SP6啟動子的重組質粒為模板制備，也可以用含啟動子的PCR產物為模板制備，本文介紹后者。（1）設計含T7啟動子的PCR引物由于PCR產物將作為合成反義RNA的模板，所以一對引物中的下游引物5’-端要含T7啟動子序列: T7啟動子......閱讀全文
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