single stranded m13mp18  (New England Biolabs)

Journal: Molecular Biology and Evolution

Article Title: Phylogenetic and functional characterization of Asgard primases

doi: 10.1093/molbev/msaf330

Figure Lengend Snippet: Primer synthesis by B18_G1 primase. a) Primer synthesis on various templates. Protein was incubated with indicated templates (1 μM for SP2 or dT35 and 230 ng for M13mp18 ssDNA), 10 μM dNTPs (1 μCi [α- 32 P]dATP) and 100 μM rNTPs in the standard assay mixture for 30 min at 55 °C. b) Primer synthesis at the indicated temperatures. Protein (1.5 μM) was incubated with M13mp18 ssDNA (230 ng), 10 μM dNTPs (1 μCi [α- 32 P]dATP), and 100 μM rNTPs in the standard assay mixture for 30 min at the indicated temperatures. c) Comparison of primases from Pyrococcus furiosus (pfu), Sulfolobus solfataricus P2 ( Sso ), and Gerdarchaeota B18_G1 (Gerd) in priming synthesis on different substrates. Reactions were performed at 55 °C for Gerd_ePriSL and Sso _PriSLX or 75 °C for pfu _PriSL.

Article Snippet: The labeled template was prepared by annealing a labeled primer (D25 or R25, ) with M13mp18 ssDNA (New England Biolabs, USA) at a 1:1.25 molar ratio.

Techniques: Incubation, Comparison

Journal: Molecular Biology and Evolution

Article Title: Phylogenetic and functional characterization of Asgard primases

doi: 10.1093/molbev/msaf330

Figure Lengend Snippet: Primer extension by B18_G1 primase and DNA binding by Gerd_ePriS. a) Primer extension by B18_G1 primase. The reaction mixture containing Gerd_ePriS or Gerd_ePriSL (1.5μM), 4 nM 32 P-labeled D25 annealed to M13mp18 ssDNA, 10 μM dNTPs or 10 μM rNTPs, 50 mM MES-NaOH, pH 7.0, 100 μg/mL BSA, and 10 mM MnCl 2 was incubated at 55 °C for 30 min. Reactions were stopped by the addition of SDS (0.8%) and protease K (1.6 mg/mL). The products were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, and analyzed on 15% polyacrylamide gel (19:1) containing 8 M urea. The gel was exposed to X-ray film. b) DNA binding by Gerd_ePriS. Gerd_ePriS at indicated concentrations were mixed with 4 nM 32 P-labeled ssDNA (D25, ).

Article Snippet: The labeled template was prepared by annealing a labeled primer (D25 or R25, ) with M13mp18 ssDNA (New England Biolabs, USA) at a 1:1.25 molar ratio.

Techniques: Binding Assay, Labeling, Incubation