一种从RNA Blot杂交膜上脱除探针的改进方法  

An Improved Method for Stripping the Probes from the Membrane Blotting RNAs after Hybridization

作　　者：姚志海 叶志华[2] 梁建庆[2] 张向阳 柳海林 杜纲国 高冬梅 

机构地区：[1]深圳市君轩生物技术有限公司,广东深圳518029 [2]广东省微生物研究所广东省菌种保藏与应用重点实验室,广东广州510070

出　　处：《生物技术》2004年第4期28-30,共3页Biotechnology

基　　金：广东省科技攻关项目 (2KM0 2 50 4G0 )

摘　　要：目的 :探讨脱除RNABlot膜上的杂交探针的方法 ,解决杂交中RNABlot膜重复使用的问题。方法 :经NBT BCIP化学显色的RNABlot杂交膜先用二甲基甲酰胺在 6 0℃处理 90min ,然后用脱探针溶液 (5 0 %去离子甲酰胺、5 0mmol LTris -HClpH7 5、5 %SDS)在 80℃处理 90min ,经过处理的膜再次用于下一轮的杂交。结果 :用这一改进的方法 ,可以完全脱除杂交膜上的地高辛标记的cDNA探针 ,RNABlot膜可以重复杂交 5轮以上 ,杂交带仍然保持清晰锐利。结论 :该改进方法操作简便 。Objective:To establish the improved method for stripping and reprobing of the membrane blotting RNAs in the hybridization.Methods:After the immunological detection with Anti-Digoxigenin-AP conjugate and NBT/BCIP in the first hybridization,the probed membrane was incubated in dimethylformamide (DMF) at 60℃ for 90 minutes with gentle shaking,until the blue color precipitates were removed from the face of the membrane by several changes of DMF.The membrane was then placed in the probe-stripping solution (50% deionized formamide,50 mmol/L Tris-HCl pH7.5,5% SDS) and treated at 80℃ for 90 minutes with gentle agitation,with replacing the probe-stripping solution twice during the incubation.Results:By Using this improved method,the DIG-labeled cDNA probes can be completely stripped,and the membrane can be then reprobed up to 5 times without significantly loss of signal.Conclusion:The improved method is convenient and effective for stripping the DIG-labeled cDNA probes from the membrane blotting RNAs after hybridization.

关 键 词：RNA Blot膜 杂交 脱除 二甲基甲酰胺 地高辛标记 CDNA探针 

分 类 号：Q781[生物学—分子生物学] Q522.6