Lichtenstein A V, Moiseev V L
Laboratory of Tumor Biochemistry, Institute of Carcinogenesis, All-Union Cancer Research Center, Moscow, Russia.
Anal Biochem. 1992 Dec;207(2):280-4. doi: 10.1016/0003-2697(92)90012-v.
We describe a technique for repeated use of 33P-labeled DNA probes in Southern hybridization experiments. A nick-translated 33P-labeled DNA probe in a volume of 0.5-1.0 ml of hybridization mixture (final concentration, 10-100 ng/ml) is used to wet a sheet of filter paper (approx 10 microliters/cm2), which covers a nylon membrane with DNA transferred by Southern blotting, and both are set between two washed X-ray films. The "sandwich" is placed in a plastic bag for hybridization for 16-24 h at 42 degrees C. This very simple procedure using 33P-labeled DNA probes has a number of advantages over the standard method using 32P-labeled probes: (a) a significantly lower biohazard (body/arms exposure); (b) a very small volume of hybridization mixture in contact with a DNA-containing membrane and the higher probe concentrations attainable, causing some increase in sensitivity, and, finally, (c) repeated use of the probe-containing filter (over approx 3 days for unique sequences and up to 2 weeks for reiterated sequences) due to a relatively long 33P half-life (25.3 days).
我们描述了一种在Southern杂交实验中重复使用³³P标记DNA探针的技术。将体积为0.5 - 1.0 ml杂交混合物（终浓度为10 - 100 ng/ml）中的缺口平移³³P标记DNA探针用于浸湿一张滤纸（约10微升/平方厘米），该滤纸覆盖着通过Southern印迹转移了DNA的尼龙膜，然后将两者置于两张冲洗过的X光片之间。“三明治”放入塑料袋中，于42℃进行16 - 24小时杂交。这种使用³³P标记DNA探针的非常简单的方法与使用³²P标记探针的标准方法相比具有许多优点：（a）生物危害显著降低（身体/手臂暴露）；（b）与含DNA膜接触的杂交混合物体积非常小，且可达到更高的探针浓度，从而使灵敏度有所提高，最后，（c）由于³³P半衰期相对较长（25.3天），含探针的滤纸可重复使用（对于单一序列约3天，对于重复序列长达2周）。
