Single-molecule imaging of RNAP-DNA interactions

Details of the experimental setup were described previously (27 (link),30 ). Briefly, an Olympus IX81 TIR microscope, equipped with a 60x oil-immersion objective (CFI APO TIRF, NA 1.49, Nikon), two lasers (532 and 640 nm; Cobolt), and a sCMOS camera (PrimeBSI; Teledyne Photometrics), was used to image RNAP–Alexa647, and DNA molecules via Sytox Orange (Thermo Fischer) intercalating dyes. Fluorescent emission was first filtered by a dichroic mirror (FF635-Di02; Semrock) and for the Sytox Orange and Alexa647 channels the band-pass filters FF01-731/137 (Semrock) and FF01-571/72 (Semrock) were used, respectively.

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Janissen R., Barth R., Polinder M., van der Torre J, & Dekker C. (2023). Single-molecule visualization of twin-supercoiled domains generated during transcription. Nucleic Acids Research, 52(4), 1677-1687.

Publication 2023

PrimeBSI Sytox Orange FF635-Di02 FF01-731/137 FF01-571/72

Corresponding Organization :

Other organizations : Delft University of Technology

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Variable analysis

independent variables

Use of Olympus IX81 TIR microscope
Use of 60x oil-immersion objective (CFI APO TIRF, NA 1.49, Nikon)
Use of two lasers (532 and 640 nm; Cobolt)
Use of sCMOS camera (PrimeBSI; Teledyne Photometrics)

dependent variables

Imaging of RNAP–Alexa647
Imaging of DNA molecules via Sytox Orange (Thermo Fischer) intercalating dyes

control variables

Dichroic mirror (FF635-Di02; Semrock)
Band-pass filters FF01-731/137 (Semrock) for Sytox Orange channel
Band-pass filters FF01-571/72 (Semrock) for Alexa647 channel

controls

No positive or negative controls were explicitly mentioned in the provided information.

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