hind iii digested λ-phage dna  (Millipore)

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