Biology
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用于下一代测序的基于磁珠的蚊子DNA提取协议
Chapters
Summary
Please note that all translations are automatically generated.
Click here for the English version.
这里描述的是一个DNA提取协议，使用磁珠从蚊子中产生高质量的DNA提取。这些提取适合下游的下一代测序方法。
Transcript
这种基于预算的磁珠DNA提取协议使资源有限的实验室和研究能够访问高通量测序。该协议不需要昂贵的设备进行高质量的DNA提取，它可以帮助某些诊断或研究的情况下，获得足够数量的DNA与高通量测序的质量是具有挑战性的。此协议很容易通过一次性演示进行复制。
应首先用少量样本进行尝试，以熟悉工作流程。首先，通过加入100微升PCR级水，在4摄氏度下孵育一小时，软化组织，使储存在70%以上酒精中的蚊子组织样本得到水合。孵化后，丢弃水，加入100微升蛋白酶K缓冲酶混合物。
然后使用微中心管虫子使组织同质化。同质化后，离心组织在56摄氏度下解压并孵育两到三个小时。为了提取DNA，移液器100微升的组织裂解成一个新的清洁微中心管。
然后添加磁珠主组合的215微升。使用移液器，将解酯和磁珠混合物混合 10 到 20 秒，然后让它在室温下站立 10 分钟。在孵化过程中，偶尔轻轻摇动管子，以最大限度地将DNA与磁珠结合。
接下来，将管子放在磁珠分离器上，直到溶液变得清晰。然后使用移液器，轻轻吸气管中的液体，而不会干扰持有DNA的磁珠。将管子从磁珠分离器移开，通过添加 325 微升洗涤缓冲器来清洗珠子。
在室温下通过管道彻底混合并孵育一分钟。孵化后，将管子放在磁珠分离器上，取出先前演示的超高纳特，然后将管子从磁珠分离器移开，用洗涤缓冲器洗涤珠子一次。第二次用缓冲器清洗后，用250微升的洗涤缓冲器两次以同样的方式清洗珠子。
最后一次洗涤后，将管子从磁珠分离器移开，并添加 100 微升的洗脱缓冲器。通过管道彻底混合，然后在室温下孵育管子两分钟，然后放回磁分离器上。当溶液变得清晰时，将超高纳特转移到一个新的，干净的0.5毫升微中心管，并适当地存储。
这里显示为典型的微卷荧光仪读数蚊子DNA在一个包含0.5毫摩尔EDTA的椭芽缓冲器。平均260至280纳米吸收比为2.3。此表显示了不同提取方法在一个工作日内处理的成本和样本数量。
本协议提取的典型试剂和消耗成本约为每个样品 9.50。此成本相当于任何典型的磁珠提取方法。本协议的主要成本效益来自不需要自动DNA提取仪器。
在处理多个样品时，请务必更改样品之间的移液器提示，以防止 DNA 交叉污染。我们使用这种方法提取的高质量DNA进行全基因组测序。我们目前正在使用测序数据来识别公共卫生和疾病控制中关注的杀虫剂耐药性或免疫基因的新突变。
拥有经济实惠的高吞吐量测序解决方案为旨在了解影响许多新基因控制策略成功的蚊子分散和基因流动的人口基因组学和景观基因组学打开了令人兴奋的大门。
Tags
生物学，第170期，DNA提取，蚊子，昆虫Related Videos
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