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Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs
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Genetics
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JoVE Journal Genetics
Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

DOI:
10.3791/55239-v

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11:24 min

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June 12, 2017

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, , ,
1Toxicology and Mycotoxin Research Unit,NPRC, USDA-ARS, 2Hazera Seeds LTD, Brurim, 3Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” – Universidad de Málaga – Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Estación Experimental “La Mayora”

Chapters

  • 00:05Title
  • 01:02Primer Design for PCR Amplification of Gene Flanks
  • 02:49Production of OSCAR Constructs
  • 05:48Agrobacterium tumefaciens Mediated Transformation (ATMT) of Fungi
  • 07:45Deletion Mutant Identification by PCR
  • 08:53Results: The OSCAR Method Generates a Gene Deletion Plasmid for Fungi in a Single Reaction
  • 10:23Conclusion

Summary

Automatic Translation

Automatic Translation

Gene deletion mutants generated through homologous recombination are the gold standard for gene function studies. The OSCAR (One Step Construction of Agrobacterium-Recombination-ready-plasmids) method for rapid generation of deletion constructs is described. Agrobacterium mediated fungal transformation follows. Finally, a PCR based confirmation method of gene deletions in fungal transformants is presented.

Tags

Keywords: Rapid Deletion ProductionFungiAgrobacteriumOSCAR Deletion ConstructsHomologous RecombinationFunctional GeneticsGene Deletion MutantsDelsGate SystemPrimer DesignPCRDeletion Confirmation

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