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Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish
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JoVE 杂志 遗传学
Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

DOI:
10.3791/64686-v

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05:55 min

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February 10, 2023

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, ,
1Department of Molecular Biosciences,The University of Texas at Austin, 2Department of Nutritional Sciences,The University of Texas at Austin

Chapters

  • 00:04Introduction
  • 00:50Fish Sperm Collection
  • 02:11DNA Extraction and Amplification
  • 03:14PCR Amplicon Separation by Gel Electrophoresis
  • 04:20Results: Mutation Analysis of Sperm
  • 05:16Conclusion

Summary

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CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

Tags

Sperm ScreeningGermline EditsZebrafishCRISPRF0IndelsGenome EditsIn Vitro FertilizationPCRGel ElectrophoresisAnesthesiaCloacaCapillary ActionSodium HydroxideThermal CyclerTris-HClPCR ReactionAgarose Gel

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