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Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1
JoVE Journal
Genetics
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JoVE Journal Genetics
Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

DOI:
10.3791/60045-v

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11:27 min

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September 18, 2019

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, , , , , , , ,
1Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division (BESE),King Abdullah University of Science and Technology (KAUST)

Chapters

  • 00:04Title
  • 01:22Flow Cell Preparation
  • 03:01Single-Molecule Förster Resonance Energy Transfer (smFRET) Experiments
  • 06:25Sm-FRET Data Analysis
  • 07:10Two-Color Alternating Excitation (ALEX) FRET Experiment and Analysis
  • 08:24Results: Representative Real-Time HJ Resolution Investigation
  • 10:13Conclusion

Summary

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Presented here is a protocol for performing single-molecule Förster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

Tags

Keywords: Single-moleculeFörster Resonance Energy Transfer (FRET)Holliday JunctionGEN1Real-time InvestigationEnzymatic SystemsSingle-molecule MethodsBiomolecular InteractionsOptical And Force MeasurementsSingle-channel Flow CellQuartz SlideDouble Adhesive SheetPolytetrafluoroethylene TweezersPolyethylene TubingEpoxy GlueAvidinPBS

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