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切胶回收的注意事项

发布: 2016-02-16 842阅读 151评论

在分子克隆里，大家不要小看了切胶回收（gel extraction）这一步骤，过低的DNA浓度和质量往往会直接影响后续实验的成功与否。为了避免这种不必要的事情发生，小编在此总结了一些切胶回收时的注意事项，希望能够帮助到大家。

  1. 电泳的缓冲液 (runningbuffer) 和胶(gel) 都应该是新做的，切胶的台子也要用乙醇清理干净。最好使用一次性切胶刀片，或是将刀片洗净灭菌。不要觉得小题大做，这样才可以保证切下的DNA目的条带没有外源DNA的污染。

  2. 切胶就是要把整个目的片断所在位置的胶全部回收，所以大家通常都是在条带四周划掉，然后回收。但是不要忘了胶的厚度，正面切完还要把胶立起来，再尽量切掉前后多余的部分。为了减少胶的体积，大家也可以用相对比较薄的胶来做，薄而宽的梳子来弥补点样体积不够的问题。

  3. 减少紫外线（UV light）对胶的照射时间。如果要同时切几个目的条带的话，大家可以先把胶在日光灯下分开，然后放在紫外光下逐个切掉。通常紫外光有两个波长可以选择，建议大家选择波长较长的，可以相对减少对DNA的损伤。

  4. 根据小编的个人经验，如果操作熟练，UV曝光时间短的话，DNA的序列不会发生任何变化。但是如果你还是不放心的话，也可以考虑买肉眼可见的染色剂，例如结晶紫（crystal violet）或亚甲蓝（methylene blue）。但是这些染剂与DNA的结合能力相对比较低，敏感度差，所以只能显示出大约0.5µg以上的DNA。如此一来，DNA marker就很难被染色，也就不能判断、确定目标条带的位置。在这种情况下，大家可以先跑胶，将有DNAmarker的一部分切开放到更强的染色剂里（Ethidium bromide, GelRed等），另一部分带有目的条带的放在“安全”染剂里，确定好位置，再进行切胶回收。

  5. 如果大家觉得第4条的方法有些麻烦，还想回归简单的切胶，那么也可以考虑使用一些小技巧来加快切胶的速度。例如，Gene Catcher Tips就是一个新兴的小工具，放在pipette上一吸就可以轻松把胶切下来。大家看下图就自然明白了。小编自己还没有机会尝试，也欢迎大家分享使用心得。
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  6. 相信大多数的人都在使用一些商用的gel extraction kit来回收胶。除了要遵循实验手册，大家还需要用多花一点心思来保证DNA的质量和产量。即使实验手册里没说，也可以多加一步清洗膜的步骤(washing step)，这样可以进一步去除膜上离液盐的残留物（chaotropic salt residue）。否则这些残留物会影响接下来实验中连接酶（ligase）的效率。用乙醇清洗的时候，记得等到乙醇全部挥发之后，再进行洗脱（elution）。想知道乙醇是否污染了样品也很简单，可以尝试重新在胶上点样，如果在加入了loading dye之后，样品还是浮起来了，证明有乙醇污染。

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