Journal: Analytical chemistry

Article Title: Versatile Analysis of DNA–Biomolecule Interactions in Solution by Hydrodynamic Separation and Single Molecule Detection

doi: 10.1021/acs.analchem.8b04733

Figure Lengend Snippet: Single molecule chromatograms demonstrate the effect of conformational changes on hydrodynamic separation. All separations were performed in the same 5 μm diameter, 100 cm length capillary and were completed in less than 8 min. (a) Lambda DNA (48.5 kbp) separated in a low ionic strength buffer (20 mM EB). The raw data (black) was fit to a bigaussian peak (blue). (b) In the presence of 100 μM SPD, the mobility of Lambda DNA significantly decreased (black is raw data, red is fitted). (c) Separation of HindIII digested Lambda DNA ladder in 20 mM EB (black is raw data, green is fitted). The mobility of the smallest fragment (564 bp) most closely overlaps with the mobility of the SPD-condensed Lambda DNA. All chromatograms were fit to a series of Gaussian or bigaussian peaks and normalized by the free dye elution time. The shaded region around each peak center defines 95% of the peak area (±2 standard deviations).

Article Snippet: Lambda DNA (New England Biolabs) and Hin dIII Digested Lambda DNA (New England Biolabs) were stained with TOTO-3 iodide (ThermoFisher Scientific) at 10 ng/ μ L of DNA and 1 μ M of dye.

Techniques: Lambda DNA Preparation

Journal: Analytical chemistry

Article Title: Versatile Analysis of DNA–Biomolecule Interactions in Solution by Hydrodynamic Separation and Single Molecule Detection

doi: 10.1021/acs.analchem.8b04733

Figure Lengend Snippet: Single molecule burst shapes provide further insight into molecular properties of DNA fragments. (a) Each single molecule burst was characterized by its size (burst area) and packing density (ratio of burst height to burst width). (b) Raw fluorescence data trace of hundreds of single molecule bursts detected during the separation of the condensed lambda DNA in 100 μM SPD. (c) Burst size frequency histograms fit to a log-normal distribution with free-coiled lambda DNA (red) and SPD-condensed DNA (blue) maintaining a higher frequency of large burst sizes than free-coiled 0.564 kbp fragments (green). (d) Packing density histograms also exhibit a log-normal distribution with both free-coiled species averaging smaller packing densities than the SPD-condensed lambda DNA.

Article Snippet: Lambda DNA (New England Biolabs) and Hin dIII Digested Lambda DNA (New England Biolabs) were stained with TOTO-3 iodide (ThermoFisher Scientific) at 10 ng/ μ L of DNA and 1 μ M of dye.

Techniques: Fluorescence, Lambda DNA Preparation

Journal: Analytical chemistry

Article Title: Versatile Analysis of DNA–Biomolecule Interactions in Solution by Hydrodynamic Separation and Single Molecule Detection

doi: 10.1021/acs.analchem.8b04733

Figure Lengend Snippet: Average species mobility and single molecule burst parameters can distinguish fragment size and conformation. (a) Average relative mobility, (b) burst size geometric mean, and (c) packing density geometric mean of free-coiled 48.5 kbp lambda DNA molecules (red), SPD-condensed lambda DNA (blue), and free-coiled 0.564 kbp fragments (green). Each parameter distinguishes one molecular population by differences in effective size in solution, total DNA content, and DNA conformation. Bar values are the average of three separation data sets, and errors bars are ±1 standard deviation.

Article Snippet: Lambda DNA (New England Biolabs) and Hin dIII Digested Lambda DNA (New England Biolabs) were stained with TOTO-3 iodide (ThermoFisher Scientific) at 10 ng/ μ L of DNA and 1 μ M of dye.

Techniques: Lambda DNA Preparation, Standard Deviation

Journal:

Article Title: Capillary DNA Sequencing: Maximizing the Sequence Output

doi:

Figure Lengend Snippet: A representative agarose gel analysis of RCA-generated DNA templates. Lanes 2 thru 49 are RCA templates (from half of a 96-well plate), while lanes 1 and 51 contain 170 ng of a lambda DNA-Hind III digest (Fisher Scientific, Fair Lawn, NJ). Lane 50 is a blank (bl) lane. Five microliters of diluted (1:5) RCA products was loaded in each well of a 0.6% agarose gel, run at 130 V for 20 min, and stained with ethidium bromide. Arrows point to some templates that have lower DNA concentrations compared with the majority of the samples shown.

Article Snippet: Lanes 2 thru 49 are RCA templates (from half of a 96-well plate), while lanes 1 and 51 contain 170 ng of a lambda DNA-Hind III digest (Fisher Scientific, Fair Lawn, NJ).

Techniques: Agarose Gel Electrophoresis, Generated, Lambda DNA Preparation, Staining