Lee H, Birren B, Lai E
Department of Pharmacology, University of North Carolina, Chapel Hill 27599-7365.
Anal Biochem. 1991 Nov 15;199(1):29-34. doi: 10.1016/0003-2697(91)90265-u.
Large DNA molecules separated in pulsed-field gels are not efficiently transferred from the gel for Southern hybridization. Various procedures for fragmenting the DNA prior to transfer are in use, but quantitative details that permit reproducible application have not been reported. We have determined the optimum level of energy for uv nicking of large DNA needed to promote efficient Southern transfer and detection by hybridization. To ensure consistent results we have used a uv oven equipped with a detector that measures only 200-400 nm wavelengths, and we report the total energy delivered. Using uv nicking and the transfer techniques described, we can obtain hybridization signals overnight with single-copy DNA probes on Southern blots of large DNA fragments separated by pulsed-field gel electrophoresis.
在脉冲场凝胶中分离出的大型DNA分子，不能有效地从凝胶转移用于Southern杂交。目前使用了各种在转移前切割DNA片段的方法，但尚未报道能实现可重复应用的定量细节。我们已经确定了大型DNA进行紫外线切口所需的最佳能量水平，以促进有效的Southern转移和杂交检测。为确保结果的一致性，我们使用了配备仅能测量200 - 400纳米波长的探测器的紫外线烤箱，并报告了传递的总能量。使用紫外线切口和所述的转移技术，我们可以在通过脉冲场凝胶电泳分离的大型DNA片段的Southern印迹上，用单拷贝DNA探针在一夜之间获得杂交信号。
