2 log dna ladder

Extension reactions of the fluorescent-labeled 32-mer primer 5′-Cy5-TGCCAAGCTTGCATGCCTGCAGGTCGACTCTA-3′ annealed to the single-stranded circular M13mp18 template (7 nM) were performed in 12.5 µl of 50 mM Tris (pH 8.0), 1 mM dithiothreitol, 50 mM NaCl, 5 mM MgCl₂, and 200 µM each of dNTPs in the presence or absence of PCNA at the indicated concentrations. DNA polymerization was initiated by addition of DNAPs (25 nM) and was conducted at 60 °C for 30 min. Reactions were quenched on ice by addition of one volume of 90% deionized formamide and 20 mM EDTA, before heating at 95 °C for 5 min. Products were resolved on 1% (w/v) alkaline agarose gel. DNA markers (2-Log DNA ladders, New England Biolabs) were loading in 45% deionized formamide and 10 mM EDTA, and run into the same gel at 4 °C for 14 h 30 min at 30 V. After electrophoresis, gels were stained with SYBR Gold (Invitrogen). Gels were first scanned with a Mode Imager Typhoon 9500 (GE Healthcare) for Cy5 to visualize the Cy5-labeled products and then scanned for SYBR Gold for detecting the ladders. Full-length 7249 bp (%) corresponds to the intensity of 7249 bp bands as a percentage of total lane intensity. In all cases, the background value was subtracted. The mean of percentage ± SD of full-length 7249 bp (%) from three independent experiments were obtained (the raw data are provided in Supplementary Table 2).

5′Fam L93 (25 nM) was first incubated with 0.8 µM RadA for 10 min at 65 °C in a buffer containing 20 mM Tris-HCl, pH 8.0, 10 mM DTT, 50 µg/mL BSA, 10 mM MgCl₂, 0.2 mM dNTPs and 2.5 mM ATP (when indicated). Then, 25 nM of purified pUC19 was added and incubated for another 10 min. Next, the reaction was mixed with DNA polymerases at indicated concentration for a further 60 min. PCNA was added to the reaction mixture when indicated at 675 nM. The final reaction volume was 20 µL, containing 130 mM NaCl. DNA products were separated either by native agarose gel (as described in D-loop formation assays) or by denaturing gels. For denaturing gel electrophoresis, the reactions were terminated by addition of 86% deionized formamid, 0.01 N NaOH and 10 mM EDTA with equal volume. DNA products were analyzed by 5% denaturing polyacrylamide gel (8 M urea) or by 1% alkaline agarose gel. The electrophoresis images were obtained and the products were quantified as described above. The alkaline agarose gel was stained with SYBR Gold, scanned first to visualize HL-647-labeled products and then scanned for SYBR Gold for detecting the ladder (2-Log DNA ladders, New England Biolabs, Ipswich, MA, USA) and the plasmid pUC19.