DNA送测序,查找目标片段的存在和准确性,已经是分子生物实验中常见的不能再常见的步骤了。但总有那么一两次,我们送测序后得到的结果是非常低质量的。这里所谓的低质量,即指,测序长度过短、不确定碱基过多(各种N飞来飞去)、重复性低等等……这让满怀希望并且赶着实验进度的我们有苦说不出,当然我们自然可以要求测序公司进行重测,但有时候,我们再次拿到的结果往往还是低质量的。如果遇到此种情况,还是重新准备自己的质粒,拿新样本再送去测序吧~
那么问题到底出在哪里呢?为什么会得到低质量的测序结果,难道测序公司的机器还会有心情不好的时候,故意给你吐个差结果。多数(不是全部哦~)测序公司的测序设备是没有问题的,一般情况下跑一次测序就可以得到有质量的结果;那么最主要的问题应该出在我们送检的样本或者引物上。
BioEngX小编在这里总结了8点注意,供大家参考。如果能够做到这八点要求,应该可以保证每次得到高质量的测序结果。
1Miniprep不敷衍
如果我们送测序的样本是质粒,那么质粒的质量是关键,这也就牵扯到我们如何有效地提取质粒。一份可适用于测序的质粒样本应该是没有细胞基因组DNA,没有RNA,也没有多余的盐的!
值得庆幸的是,现在市面上出售的各种miniprep试剂盒,都有非常详细的操作说明,请一步一步明确按照指示来操作。该裂解4分钟的,千万别裂解10分钟;该离心几次,千万别少;洗出时该静置的,要静置……
想了解miniprep原理及具体操作方法,请戳这里:质粒提取,你不知道的事~
2干净的PCR样本
如果你送检的样本是PCR样本,那么记得纯化以后再送检。PCR反应残留液中的盐,会影响到后续测序结果的。
3样本质量大检查
我们自己能够做到的DNA样本质量检测无外乎两种:要么是紫外分光光度计测浓度;要么是琼脂糖胶分离样本看纯度。
紫外分光光度计,可以告诉我们样本DNA的浓度,如果波谱上同时出现非260nm波峰的话,证明有其他杂质(蛋白或其他,取决于峰的位置)。琼脂糖胶可以帮助分离样本中不同长度的DNA,如果质粒样本中有其他杂质DNA(如,基因组DNA),我们可以很清楚地从条带的高低看出来。万一样本中有大量杂质DNA,那只能重新提取质粒样本了。
4了解送检样本的要求
每个测序公司也许对样本的浓度和体积要求不一样,所以千万查看清楚,这家公司需要多少微升多少浓度的什么样本。如果你的原始样本浓度过高,请一定稀释后再送检。
5准确的引物
一般测序公司里会存储各种常见引物,比如T7F、M13R、SP6等等……但是每家公司的标准引物不一定完全一样。在送检前,请一定检查测序公司的引物序列具体是什么,是否和自己的送检DNA上的引物片段吻合。
如果是随样本送检的DIY引物,一般要满足以下几点要求:
- Tm保持在50-60 °C
- 避免二级结构和可能的自身配对联合
- 长度保持在18-24 bp
- 溶解于dH2O,并配到测序公司指定的浓度
- 引物重合位置应与目标片段起始位置相隔50 bp左右
6阳性对照
如果你有已经被测过序并且得到高质量结果的样本的话,可以把这个样本与其他未知样本同批次送检,这样就可以保证在那一批次的测序中有一个阳性对照(positive control)。这样我们就能清楚地知道测序机器是否正常工作。
7二次测序
当第一次测序得到了垃圾结果,我们可以要求测序公司再对自己的样本进行重新测序,很多公司对因不满意第一次测序而要求的再测序是不收费的,一定要选择有这种服务的公司。一旦第一次测序不成功,自己还有第二次机会,既然花了钱就要有最大的效果。
8多家公司,多个选择
如果自己的样本在同一家公司反复测得垃圾结果,而且自己的样本是确保没有问题的,那么换一家测序公司吧,你们八字不合,测不出来的~~~
这就是BioEngX小编认为,能获得高质量测序结果应该注意的8件事,欢迎各位关于测序的别的想法~在文末留言哦~
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