Liou J T, Shieh B H, Chen S W, Li C
Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan, Taiwan, ROC.
Prep Biochem Biotechnol. 1999 Feb;29(1):49-54. doi: 10.1080/10826069908544692.
This study is to improve the digestion pattern of miniprepped plasmid analyzed on gel. Frequently, some ambiguous DNA bands, which are suspected to be denatured DNA molecules, appear during electrophoresis of enzyme digested miniprepped plasmids. By employing Southern hybridization of two identical gels, one had been treated with denaturation-neutralization step and another without such treatment, we confirmed that many of these ambiguous DNA bands were single-stranded (SS) DNA molecules. The presence of SS DNA was due to the use of excess amount of NaOH during plasmid DNA purification with the conventional alkaline lysis method. We, therefore, modified the procedure and recommend that a half amount of NaOH (0.1N instead of 0.2N) should be used when isolating small quantity of plasmid DNA with the method.
本研究旨在改善凝胶分析的小量制备质粒的消化模式。在酶切小量制备质粒的电泳过程中，经常会出现一些疑似变性DNA分子的模糊DNA条带。通过对两块相同凝胶进行Southern杂交，一块经过变性-中和步骤处理，另一块未经过此类处理，我们证实这些模糊DNA条带中有许多是单链（SS）DNA分子。SS DNA的存在是由于在使用传统碱性裂解方法纯化质粒DNA过程中使用了过量的NaOH。因此，我们修改了该程序，并建议在用该方法分离少量质粒DNA时，应使用半量的NaOH（0.1N而非0.2N）。
