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定点突变常见问题及对策

定点突变并不是一个复杂的实验(原理可见文章定点突变简说),然而有时候我们在实验中却会遇到各种各样的问题。一些常见的问题包括但不限于: 菌落太多;没有菌落生长;得到菌落,但菌落中不含有目的突变点等。如何分析出现问题的原因并解决这些问题呢?小编能提供的最好的建议就是在进行定点突变实验时一定要有对照组(control)。许多定点突变试剂盒中都会包含对照用的模板,引物以及转化时候的标准质粒。对照实验能够帮助你更容易地确定可能出现问题的实验操作步骤,节省你的时间以及试剂耗材。

下面是一些在定点突变实验过程中解决常遇问题的小技巧:

当平板上菌落(colonies)太多时

  • 降低PCR反应中的模板(template)浓度
  • 减少转化时PCR产物的用量
  • 将不同体积的转化后产物涂在平板上,比如10 µl, 20 µl, 50 µl, 100 µl。选取菌落分布最合适(well-spaced)的平板挑选阳性克隆。
  • 延长Dpn I酶切(digestion)时间,比如酶切2个小时,代替1个小时

当平板上没有菌落时

  • 提高PCR反应中模板的用量
  • 提高转化时PCR 产物的用量
  • 如果DNA电泳中显示PCR 产物浓度不高或PCR失败,可以试用不同的温度梯度进行PCR 反应
  • 改变PCR反应的延伸时间及温度(比如降低温度到68 oC, 以60 s /Kb的比例设置延伸时间)
  • PCR反应时加入一点 DMSO (2-8%),用来辅助富含GC的区域双链的分离
  • 检查感受态(competent cells)细胞是否有效(利用对照质粒)
  • 浓缩酶切后的DNA,可以利用乙醇(Ethanol)沉淀(precipitate)酶切过的DNA, 然后在转化前用更小体积的液体重悬(re-suspend)之
  • 转化前纯化DNA样品,取出PCR反应中的盐离子

菌落中没有目的突变型(desired mutation)

  • 利用含有Dam甲基化酶(methylase)的菌株扩增模板质粒,比如JM109,DH5ɑ。可能有些同学不明白这为何意,待小编一一道来。Dam甲基化酶可将GATC序列中的腺嘌呤N6位点进行甲基化修饰,而这一位点恰恰是DpnI 酶的识别位点。因此不能应用Dam甲基化酶缺失的菌株生产模板质粒,比如JM110 菌株。这种菌株产生的DNA无法被DpnI酶切掉,转化DNA中含有野生型模板,将产生大量的假阳性(fake positives)。
  • 提高DpnI 酶切时间或者提高DpnI酶的用量
  • 减少PCR循环数,过多的PCR循环可能导致非特异性突变点的引入
  • 如果上述策略仍不能解决问题,那只能重新设计引物了,有关引物设计问题,见历史文章

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作者: 于浩然

本科及硕士毕业于天津大学,博士毕业于伦敦大学学院。现就职于浙江大学化学工程与生物工程学院,PI,博导。研究方向为蛋白质工程、生物催化剂、合成生物学等。



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  19. 老师,您好,想请问一下就是利用引物直接环扩质粒后用DpnI酶消化后转大肠的BL21感受态,板子长菌但是菌落PCR没有目的条带是为什么呢,然后就是用无缝克隆酶处理了一下DpnI酶消化后的产物菌P有条带,但是突变位点有很多片段插入,阳性率也低。想请问一下有解决办法吗,谢谢老师!

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