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05:30 min
September 10th, 2021
DOI :
10.3791/63008-v
Chapters
0:05
Introduction
0:33
A Gradient PCR Using Genomic DNA (gDNA) from a Single Individual
1:09
Preparation of Genomic DNA from Mosquito Legs
2:31
Perform PCR
3:35
Results: Detection of CRISPR/Cas9-Induced Indels and the Establishment of Mutant Lines in the Mosquito Aedes aegypti
5:02
Conclusion
Transcript
该协议是一种非常简单有效的方法来检测由CRISPR / Cas9技术从众多个体中产生的突变体。这种技术的两个主要优点是它可以在几个小时内完成,并且适用于多种生物体。引物设计对某些基因来说可能具有挑战性,并且可能需要多轮引物设计和验证才能使HRMA高效。
要进行梯度PCR,请先制备预混液。然后在96孔板中除去19微升用于非模板对照或NTC的预混液。然后,将模板添加到剩余的预混音中。
将20微升样品等分到96孔板中。根据循环参数执行PCR,然后使用手稿中提到的参数生成热
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Summary
本文详细介绍了快速鉴定CRISPR / Cas9诱导的内嵌的方案,以及使用高分辨率熔体分析选择蚊子 埃及伊蚊 中的突变系。
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