phage λ dna hin diii digest  (New England Biolabs)

Journal: Structure(London, England:1993)

Article Title: Structural Basis for the Activation and Target Site Specificity of CDC7 Kinase

doi: 10.1016/j.str.2020.05.010

Figure Lengend Snippet: Design and Activity of CDC7 Deletion Constructs (A) Schematic of human CDC7 and DBF4. Locations of kinase inserts (KI-2 and KI-3) in CDC7 and conserved motifs in DBF4 (N, M, and C) are indicated. Deletions in CDC7 (Δ1–36, Δ228–345, and Δ467–533) and the span of the DBF4 fragment used for crystallography in this work are shown above the schematics. (B) In vitro kinase activities of WT full-length or deleted CDC7 constructs produced in complex with DBF4 MC with or without treatment with λ phage protein phosphatase (λPPase). Activities were normalized to WT kinase pre-treated with phosphatase. The bar plots show mean values with standard deviations from triplicate measurements. (C) Effects of deletions in KI-2 and KI-3 on in vitro kinase activities of CDC7(Δ1–36)-DBF4 MC . The KI-2 deletion used for crystallography previously ( Hughes et al., 2012 ) is Δ228–359; the rightmost bar reports relative activity of the construct used for crystallography in the current work. All constructs were pre-treated with λPPase, and activities were normalized to the construct with full-length inserts. Error bars represent standard deviations based on measurements done in triplicate.

Article Snippet: DNA fragment encoding λ phage protein phosphatase ( ) was PCR-amplified using primers 5′-TGCGCTATTACGAAAAA ATTGATGG and 5′-GCGCGTCGACTGCGCCTTCTCCCTGTACC and λ phage Hin dIII digest DNA ladder (New England Biolabs) as template and cloned between Nde I and Sal I sites of pET20b(+) vector (Novagen), resulting in pET-λPPase-His6.

Techniques: Activity Assay, Construct, In Vitro, Produced

Journal: Structure(London, England:1993)

Article Title: Structural Basis for the Activation and Target Site Specificity of CDC7 Kinase

doi: 10.1016/j.str.2020.05.010

Figure Lengend Snippet:

Article Snippet: DNA fragment encoding λ phage protein phosphatase ( ) was PCR-amplified using primers 5′-TGCGCTATTACGAAAAA ATTGATGG and 5′-GCGCGTCGACTGCGCCTTCTCCCTGTACC and λ phage Hin dIII digest DNA ladder (New England Biolabs) as template and cloned between Nde I and Sal I sites of pET20b(+) vector (Novagen), resulting in pET-λPPase-His6.

Techniques: Recombinant, Software

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