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经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序
JoVE Journal
Biology
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JoVE Journal Biology
A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
DOI:

11:36 min

July 03, 2016

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Chapters

  • 00:05Title
  • 00:43Synthesis of NNS Sub-libraries for Each Amino Acid Position by Two-step PCR Site-directed Mutagenesis
  • 01:53Cloning of NNS Sub-libraries into Selection Vectors
  • 03:47Selection of the TEM-1 Whole-Protein Saturation Mutagenesis Library for Antibiotic Resistance
  • 05:51High Throughput Sequencing to Determine Fitness Effects of Mutations
  • 08:44Results: High Throughput Sequencing from Whole-Protein Saturation Mutagenesis
  • 10:16Conclusion

Summary

Automatic Translation

我们提出了一个协议为利用高通量测序蛋白的综合单站点饱和突变库功能评估。重要的是,这种方法采用正交引物对复图书馆建设和测序。提供了使用在氨苄青霉素的临床相关剂量选择的TE​​M-1β内酰胺酶代表性结果。

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