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电泳缓冲液TAE与TBE，哪个更好？

发布: 2017-05-01 2,590阅读 329评论
我们在进行DNA电泳时都会加入缓冲溶液，常用的缓冲溶液有TAE，含有三羟基氨基甲烷 Tris，乙酸acetate和EDTA 以及TBE（ Tris-硼酸borate-EDTA ）。

 

缓冲液的作用是什么？
缓冲液的作用之一是维持溶液的pH稳定。 电流通过水时，在阳极发生氧化反应，生成氧气以及氢离子；在阴极发生还原反应，生成氢气以及氢氧根离子。长时间电泳将会使阳极变酸，阴极变碱。一个好的缓冲系统应该有较强的缓冲能力，使溶液两极的pH保持基本稳定。

 

电泳缓冲液的另一个作用是使溶液带有一定的导电性，以利于DNA分子的迁移。 DNA为碱性物质，在电泳（缓冲液pH=8）时带负电荷，由负极向正极迁移。

 

电泳缓冲液还有一个组分是EDTA，目的是螯合Mg2+等离子，防止电泳时激活DNA酶。由于核酸酶需要这些离子，EDTA可以将这些离子牢牢抓住，避免核酸降解。但要注意的是，镁离子同时是很多酵素的辅助因子，如限制酶，DNA聚合酶等等，因此EDTA的浓度一般不会太高（通常在1mM左右）。

 

TAE or TBE哪个更好？
DNA电泳时应该用哪个？这其实取决于你的实验目的。下面我们来看看二者的区别[1]。
  •  TBE的电导率大于TAE的电导率。因此相比于TAE，TBE不太会导致电泳槽内过热的情况发生，长时间电泳推荐使用TBE
  • 硼酸是酶的抑制剂，因此如果你需要分离电泳的DNA用于酶切反应，建议使用TAE作为电泳缓冲溶液
  • TBE对于较小片段的分离效果更好。对于小于300bp的片段，在2%的琼脂糖凝胶上，TBE中的迁移速度更快
  • TAE适用于大片段的分离，大于2kb的片段在TAE中的迁移速度更快（0.8%的琼脂糖凝胶中），速度比在TBE中快出约10%
  • TAE更适用于回收DNA片段。
  • 相比于TBE，TAE对于超螺旋的DNA分辨率更高[2]

 

TAE与TBE的配方
在本文的最后，我们列出TAE与TBE的配方以及配置方法，供大家查询使用[3]。
TAE缓冲液
实验之中经常将TAE配制成10× 或者 50× 的储液，实验前稀释储液到1×的工作液。 1x TAE 缓冲液的成分是：
  • 40 mM Tris (pH 7.6)
  • 20 mM acetic acid
  • 1 mM EDTA
50x TAE储液配制方法：
将Tris(FW=121) 242g，100 ml 0.5 M EDTA， 57.1 ml的冰醋酸溶解在600 ml的去离子水中；而后调节pH至8.3，加去离子水定容至1L后，室温保存。
使用时稀释50倍 即1×TAE Buffer

TBE缓冲液
TBE缓冲液可以配置成 5× or 10×的储液。1x TBE 缓冲液的成分是：
  • 89 mM Tris (pH 7.6)
  • 89 mM boric acid
  • 2 mM EDTA
10x TBE 储液配制方法：
将Tris（FW=121）108g，硼酸（FW=61.8）55g，40 ml 0.5 M EDTA 溶解在600 ml的去离子水中；而后调节pH至8.3，加去离子水定容至1L后，室温保存。
使用时稀释10倍 即1×TBE Buffer

 

 参考文献
[1]三浦裕, 和気弘明, 加藤泰治. TBE, or not TBE; that is the question: Beneficial usage of tris-borate for obtaining a higher resolution of small DNA fragments by agarose gel electrophoresis[J]. Nagoya medical journal, 1999, 43(1): 1-6.
[2]Dingman C W, Peacock A C. Analytical studies on nuclear ribonucleic acid using polyacrylamide gel electrophoresis[J]. Biochemistry, 1968, 7(2): 659-668.
[3]http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6205.pdf
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