Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels
Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels
复制标题
从琼脂糖凝胶中分离和纯化大 DNA 限制性片段
DOI：
10.1002/0471142727.mb0206s59
复制
发表时间：
2002
影响因子：
--
通讯作者：
R. Ribaudo
中科院分区：
文献类型：
--
作者：
D. Moore;D. Dowhan;J. Chory;R. Ribaudo
本单元描述了从琼脂糖凝胶中恢复和纯化DNA限制性片段的方法。第一种方案描述使用缓冲填充的透析袋从标准琼脂糖凝胶中电洗脱感兴趣的片段，然后使用Elutip柱进行浓缩和纯化。这种方法可以有效地用于从50到20,000 bp的所有大小的片段。直接在NA‐45纸上电泳（第二种方案）对< 2000bp的片段提供了相对较高的收率。> 1000bp的片段也可以在低凝胶/熔融琼脂糖凝胶上分离，并通过苯酚提取（第三种方案）、b -琼脂酶消化凝胶（第一种备选方案）或通过玻璃珠提取（第二种备选方案）进行纯化。描述了使用柱而不是凝胶从片段中去除连接体，然后是估计溶液中DNA浓度的方法。
This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first protocol describes electroelution of the fragment of interest from standard agarose gels using buffer‐filled dialysis bags, followed by concentration and purification using an Elutip column. This approach can be used effectively for fragments of all sizes from 50 to 20,000 bp. Electrophoresis directly onto NA‐45 paper ( second protocol ) provides relatively high yields for fragments <2000 bp. Fragments >1000 bp can also be separated on low gelling/melting agarose gels and purified by phenol extraction ( third protocol ), b‐agarase digestion of the gel (first alternate protocol ), or via glass beads extraction (second alternate protocol ). Removing linkers from a fragment using a column rather than a gel is described, followed by a method for estimating DNA concentrations in solution.