基于尼龙膜的点杂交条件的建立及优化

Establishment and Optimization of Dot Blot Technique

目的:进行基于尼龙膜的点杂交条件的建立及优化。方法:采用带正电荷的尼龙膜对斑点杂交的每一步骤进行优化,包括预杂交液,杂交液,洗涤和封闭液等及各步反应时间,并采用最终确立的条件检测糖基转移酶基因多肽:N-乙酰氨基半乳糖转移酶2(pp-Gal NAcT2)在K562、NB4、NI H3T3和SGC7901细胞株中的差异表达以验证膜杂交条件的可行性。结果:建立并优化了膜斑点杂交的条件,本底清晰,结果可靠。结论:建立的斑点杂交技术经济简便,切实可行。

Objective To establish and optimize the dot blot technique. Methods We used the positively charged nylon membranes to optimize experiments for the dot blot including prehybridization solution,hybridization solution,washing buffer,blocking solutionetal,and the times of every step, In order to validate its feasibility,we analyzed the expression of ppGalNAc-T2 in different tumor cell lines by dot blot assay using the established hybridizationsystem. Results we established and optimized the dot blot technique which had reliable results and low background. Conclusion The established dot blot technique is practicable and economical.