Reed K C, Mann D A
Nucleic Acids Res. 1985 Oct 25;13(20):7207-21. doi: 10.1093/nar/13.20.7207.
The unique properties of nylon membranes allow for dramatic improvement in the capillary transfer of DNA restriction fragments from agarose gels (Southern blotting). By using 0.4 M NaOH as the transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer. Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline solvent induces covalent fixation of DNA to the membrane. The saving in time and materials afforded by this simple modification is accompanied by a marked improvement in resolution and a ten-fold increase in sensitivity of subsequent hybridization analyses. In addition, we have found that nylon membrane completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4 Kb. This property can be utilized in the direct electrophoretic transfer of native restriction fragments from polyacrylamide gels. Exposure of DNA to ultraviolet light, either in the gel or following transfer to nylon membrane, reduces its ability to hybridize.
尼龙膜的独特特性使得从琼脂糖凝胶中进行DNA限制性片段的毛细管转移（Southern印迹法）有了显著改进。在凝胶用酸进行短暂预处理后，使用0.4M氢氧化钠作为转移溶剂，DNA在转移过程中会脱嘌呤。所有大小的片段都会被洗脱并被膜定量保留；此外，碱性溶剂会诱导DNA与膜发生共价固定。这种简单改进带来的时间和材料节省伴随着分辨率的显著提高以及后续杂交分析灵敏度提高了十倍。此外，我们发现尼龙膜在低离子强度的转移溶剂（包括蒸馏水）中能完全保留天然（和变性）DNA，尽管从凝胶中定量洗脱DNA仅限于小于4Kb的片段。这一特性可用于从聚丙烯酰胺凝胶中直接电泳转移天然限制性片段。DNA无论是在凝胶中还是转移到尼龙膜后暴露于紫外线下，都会降低其杂交能力。
