Journal: Journal of visualized experiments : JoVE

Article Title: Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

doi: 10.3791/60784

Figure Lengend Snippet: Materials

Article Snippet: ​ Name of Material/Equipment Company Catalog Number Comments/Description Alexa Fluor 488 Labeling Kit Invitrogen A30006 Bio-Spin P-6 Gel Columns Bio-Rad 7326221 Biotin Sigma-Aldrich B4501 Use as free biotin in Step 5.6 Biotin-14-dCTP AAT Bioquest 17019 BSA-Biotin Sigma-Aldrich A8549 Coverslips VWR 48393–195 No. 1 ½, 22 × 50 mm dNTP Set Invitrogen 10297018 Float Buoys for Mini Dialysis Device Thermo Scientific 69588 Klenow Fragment (3’→5’ exo-) New England BioLabs M0212S action buffer in Step 2.1.1 and 1X reaction buffer in Step 2.2.2 Lambda DNA New England BioLabs N3011S Mini Dialysis Device Thermo Scientific 69570 10K MWCO, 0.1 mL volume NE Buffer 4 New England BioLabs B7004S NHS-PEG4-Biotin Thermo Scientific 21330 Protocatechuate 3,4-Dioxygenase Sigma-Aldrich P8279 Protocatechuic acid Santa Cruz Biotechnology sc-205818 Silicone Elastomer Kit for PDMS Fabrication The Dow Chemical Company 4019862 Streptavidin Sigma-Aldrich 85878 The Blocking Solution CANDOR Bioscience 110 050 se as casein blocking solution throughout protocol Vinyl Cleanroom Tape Fisher Scientific 19-120-3217 von Willebrand Factor, Human Plasma Millipore Sigma 681300 YOYO-1 Dye AAT Bioquest 17580 0.25 mm Inner Diameter Tubing Cole-Parmer EW-06419–00 25 Gauge Needle Thomas Scientific JG2505X Open in a separate window Materials

Techniques: Labeling, Lambda DNA Preparation, Blocking Assay

Journal: Methods in enzymology

Article Title: Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases

doi: 10.1016/bs.mie.2016.09.010

Figure Lengend Snippet: Schematic representation of a multichannel flow cell used for visualization of DNA unwinding by an individual RecBCD enzyme. (A) Two-channel flow cell connected to a syringe pump. First channel contains RecBCD–DNA–bead complex and the second channel, the “reaction channel” contains ATP. “X” indicates the position of the optical trap, and this region is magnified to show movement of the enzyme-DNA–bead complex to the reaction channel. The RecBCD–DNA–bead complex is moved into the reaction channel by moving the stage by 400 μm. (B) The optically trapped fluorescently labeled dsDNA is extended by flow and its observed length decreases as RecBCD unwinds and degrades it. (C) Sequential images of DNA shortening as a function of time (t, in seconds) by RecBCD. Panels (A) and (B): From Bianco, P. R., Brewer, L. R., Corzett, M., Balhorn, R., Yeh, Y., Kowalczykowski, S. C., & Baskin, R. J. (2001). Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature, 409(6818), 374–378. doi:10.1038/35053131. Panel (C): From Liu, B., Baskin, R. J., & Kowalczykowski, S. C. (2013). DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate. Nature, 500(7463), 482–485. http://dx.doi.org/10.1038/nature12333.

Article Snippet: Mix 4.8 μL of 1.22 μ M biotinylated RecBCD enzyme (stored in 20 m M Tris–HCl (pH 7.5), 0.1 m M EDTA, 0.1 m M DTT, 100 m M NaCl, and 50%, v/v glycerol) with 3 μL of streptavidin-coated florescent nanoparticle (~18 n M ) (40 nm TransFluoSpheres; excitation 488 nm; emission 645 nm; Molecular Probes, Carlsbad, CA) in 50 m M sodium phosphate (pH 7.5), 50 m M NaCl, and 0.02% (v/v) Tween 20.

Techniques: Labeling, Translocation Assay

Journal: Methods in enzymology

Article Title: Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases

doi: 10.1016/bs.mie.2016.09.010

Figure Lengend Snippet: Direct visualization of translocation by fluorescently labeled RecBCD enzyme. (A) Illustration of an optically trapped DNA molecule with fluorescently labeled RecBCD bound to the free end. The optically trapped dsDNA is extended by flow. In presence of ATP, the RecBCD enzyme (with fluorescent nanoparticle attached) translocates along the DNA from right to left, opposite to the direction of flow. (B) Sequential images of the RecBCD–nanoparticle complex translocating on two different DNA molecules, one devoid of Chi (left) and the other containing Chi (right). The intense bright spot on the left of each frame is the streptavidin-coated polystyrene bead, which is fluorescent due to nonspecific binding of the nanoparticles. The position of the RecBCD nanoparticle (faint spot to the right) is shown by an arrow in each frame. The numbers in image are an arbitrary time in seconds. Panels (A) and (B): From Handa, N., Bianco, P. R., Baskin, R. J., & Kowalczykowski, S. C. (2005). Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after χ recognition. Molecular Cell, 17(5), 745–750. http://dx.doi.org/10.1016/j.molcel.2005.02.011.

Article Snippet: Mix 4.8 μL of 1.22 μ M biotinylated RecBCD enzyme (stored in 20 m M Tris–HCl (pH 7.5), 0.1 m M EDTA, 0.1 m M DTT, 100 m M NaCl, and 50%, v/v glycerol) with 3 μL of streptavidin-coated florescent nanoparticle (~18 n M ) (40 nm TransFluoSpheres; excitation 488 nm; emission 645 nm; Molecular Probes, Carlsbad, CA) in 50 m M sodium phosphate (pH 7.5), 50 m M NaCl, and 0.02% (v/v) Tween 20.

Techniques: Translocation Assay, Labeling, Binding Assay

Journal: Methods in enzymology

Article Title: Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases

doi: 10.1016/bs.mie.2016.09.010

Figure Lengend Snippet: Visualizing unwinding of individual DNA molecules using fluorescent SSB protein and TIRF microscopy. (A) Top: Diagram of phage λ DNA molecule with the indicated number of biotin groups incorporated in each 12-nt cos overhang. Middle: Illustration of biotinylated λ DNA attached at both ends via biotin–streptavidin linkage. Bottom: Image of an actual λ DNA molecule attached to the glass surface, stained with YO-PRO-1 (100 nM), and illuminated with a 488 nm laser. The image is false colored in green and the attachment points to the glass surface are indicated. (B) The process required to construct a flow cell containing three separate single-channels. The steps highlighted are equivalent to those described in Section 4.1. (C) The flow cell from (B) mounted onto the objective; biotinylated lambda DNA was injected under buffer flow, permitting attachment of both ends to the surface. Unwinding tracks are visualized by binding of AF488-SSBG26C (green) to ssDNA regions. (D) Schematic representation of a TIRF microscope capable of visualizing DNA unwinding by RecQ by monitoring signal from both DNA and fluorescent SSB simultaneously. As shown in (C) the flow cell is mounted onto a 100× oil-immersion objective. The fluorescent SSB and DNA are excited by two lasers; 488 and 561 nm, respectively, and emission measured, via dichroic mirrors (M1 and M2). The deconvoluted emission is then directed onto different areas of a CCD camera generating a signal corresponding to either SSB or DNA. The lasers are operated using a custom LABview VI program to coordinate excitation with image acquisition so that the sample is illuminated only during the exposure times. Panels (A) and (D): From Rad, B., Forget, A. L., Baskin, R. J., & Kowalczykowski, S. C. (2015). Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding. Proceedings of the National Academy of Sciences of the United States of America, 112(50), E6852–E6861. http://dx.doi.org/10.1073/pnas.1518028112.

Article Snippet: Mix 4.8 μL of 1.22 μ M biotinylated RecBCD enzyme (stored in 20 m M Tris–HCl (pH 7.5), 0.1 m M EDTA, 0.1 m M DTT, 100 m M NaCl, and 50%, v/v glycerol) with 3 μL of streptavidin-coated florescent nanoparticle (~18 n M ) (40 nm TransFluoSpheres; excitation 488 nm; emission 645 nm; Molecular Probes, Carlsbad, CA) in 50 m M sodium phosphate (pH 7.5), 50 m M NaCl, and 0.02% (v/v) Tween 20.

Techniques: Microscopy, Staining, Construct, Lambda DNA Preparation, Injection, Binding Assay

Journal:

Article Title: Efficient purification of DNA fragments using a protein binding membrane

doi:

Figure Lengend Snippet: Retainment of labelled lambda HindIII restricted DNA fragments by the membrane cartridge. Lambda HindIII DNA fragments are labelled with streptavidin in (A), and with fluorescein in (B). An aliquot of 1 µg lambda HindIII digested DNA fragments were used in each lane. Lane 1, unlabelled lambda HindIII fragments; lane 2, unlabelled lambda HindIII fragments incubated with 5 µg BSA and passed through the membrane cartridge; lane 3, unlabelled lambda HindIII fragments incubated with 5 µg streptavidin and passed through the membrane cartridge; lanes 4 and 5, empty; lane 6, labelled lambda HindIII fragments; lane 7, labelled lambda HindIII fragments incubated with 5 µg BSA and passed through the membrane cartridge; lane 8, labelled lambda HindIII fragments incubated with 5 µg streptavidin (A) or anti-fluorescein antibody (B) and passed through the membrane cartridge.

Article Snippet: Lambda fragment labelling Two micrograms of lambda Hin dIII digested DNA (Gibco BRL) was added 40 U of E.coli DNA polymerase I large Klenow fragment (New England Biolabs), 2 nmol of 14-modified-dCTP (biotin or fluorescein) (Gibco BRL) and 10 nmol of dGTP (Gibco BRL) giving a total volume of 100 µl.

Techniques: Incubation

Journal:

Article Title: Efficient purification of DNA fragments using a protein binding membrane

doi:

Figure Lengend Snippet: Purification of digested DNA. An aliquot of 200 ng of DNA was used for each lane. (A) Lane 1, 100 bp DNA size standard; lane 2, empty; lane 3, PCR product; lane 4, PCR product added streptavidin and passed through the membrane cartridge; lane 5, empty; lane 6, PCR product restricted by SfiI endonuclease; lane 7, PCR product restricted by SfiI endonuclease added streptavidin and passed through the membrane cartridge; lane 8, empty; lane 9, PCR product restricted by NotI endonuclease; lane 10, PCR product restricted by NotI endonuclease added streptavidin and passed through the membrane cartridge; lane 11, empty; lane 12, PCR product restricted by NotI and SfiI endonucleases; lane 13, PCR product restricted by NotI and SfiI endonucleases added streptavidin and passed through the membrane cartridge; lane 14, empty; lane 15, 100 bp DNA size standard. (B) Lane 1, Lambda HindIII size standard; lane 2, empty; lane 3, AscI digested and biotin labelled plasmid DNA; lane 4, AscI digested and biotin labelled plasmid DNA added streptavidin and passed through the membrane cartridge; lane 5, empty; lane 6, AscI digested and biotin labelled plasmid DNA restricted by SfiI endonuclease; lane 7, AscI digested and biotin labelled plasmid DNA restricted by SfiI endonuclease added streptavidin and passed through the membrane cartridge; lane 8, empty; lane 9, AscI digested and biotin labelled plasmid DNA restricted by NotI endonuclease; lane 10, AscI digested and biotin labelled plasmid DNA restricted by NotI endonuclease added streptavidin and passed through the membrane cartridge; lane 11, empty; lane 12, AscI digested and biotin labelled plasmid DNA restricted by NotI and SfiI endonucleases; lane 13, AscI digested and biotin labelled plasmid DNA restricted by NotI and SfiI endonucleases added streptavidin and passed through the membrane cartridge; lane 14, empty; lane 15, Lambda HindIII size standard.

Article Snippet: Lambda fragment labelling Two micrograms of lambda Hin dIII digested DNA (Gibco BRL) was added 40 U of E.coli DNA polymerase I large Klenow fragment (New England Biolabs), 2 nmol of 14-modified-dCTP (biotin or fluorescein) (Gibco BRL) and 10 nmol of dGTP (Gibco BRL) giving a total volume of 100 µl.

Techniques: Purification, Plasmid Preparation