Preparation of Quantum-Dot-Labeled DNA Substrate Materials
3:38
Microfluidic Device Construction
5:58
Surface Tethering of Quantum-Dot-Labeled DNA Substrate
7:07
REase-Mediated DNA Cleavage
7:51
Results: Single-Molecule TIRF Experiment for Parallelized Observations of DNA Cleavage Events
9:32
Conclusion
Transcript
Med denne protokollen kan vi direkte observere stedsspesifikk DNA-spaltning ved restriksjonsendonukleaser på enkeltmolekylnivå. Vår multipleksede tilnærming lar oss måle tiden det tar for hundrevis av individuelle REases å fullføre katalytisk sykl
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Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.