Dntps

Ten pmol of the Cre PA substrate (with RE site of interest) was incubated with 2 units purified RE or 20% reaction volume of lysate with appropriate CFU/µL in CutSmart buffer and 1 unit/µL AP for 1 h at 37 °C. Then, 50 mM EDTA and 1 unit/µL Cre recombinase was added and the circularization was performed 1 h at 37 °C and inactivated by 10 min incubation at 95 °C. The DNA circles were hybridized to the functionalized glass slides 1 h in a humidity chamber at 37 °C. The slides were washed 1 min in wash buffer 2, 1 min in wash buffer 3, and dehydrated 1 min in 96% ethanol. RCA was performed 2 h at 37 °C with 1 unit/μL Phi29 DNA polymerase in 50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2S04, 4 mM DTT pH 7.5, 0.2 μg/μL BSA, 250 μM dNTPs and 12.5 μM atto-488-dUTP (Jena Bioscience, Germany) in a humidity chamber. The reaction was stopped by washing 10 min in wash buffer 2, 5 min in wash buffer 3 and 1 min 96% ethanol. Vectashield and cover glass were added and the slide were analyzed with a fluorescent microscope (Olympus IX73) at 60× magnification. Ten pictures were acquired per sample and the number of signals were analyzed using the ImageJ software.

Each PCR reaction was conducted in a volume of 25 μL containing 10X buffer B (NEB), dNTPs (Jena Bioscience GmbH), MgCl₂ (NEB), forward and reverse primers (Sigma), and Taq polymerase (Solis Biodyne). The reaction allowed 15 min of initial denaturation followed by 35 cycles of 30 s at 95 °C for denaturation, 45 s at 60 °C for annealing, and 60 s at 72 °C for elongation. Final elongation was carried out at 72 °C for 10 min. The PCR products were run on a 1% agarose gel at 100 V for 45 min and visualized using an INTAS imager.

Reactions were assembled in buffer containing 10 mM Bis–Tris–propane–HCl (pH 7.0), 10 mM MgCl₂, and 100 μM DTT, supplemented with 2 μM PrimPol, 250 μM dNTPs, 2.5 μM FAM dNTPs (dATP, dCTP, dUTP) (Jena-Biosciences), 1 μM single-stranded template (sequence 7, Supplementary Table S1), and varying concentrations of PolDIP2 or GST (as indicated on individual figures). Reactions were incubated at 37°C for 15 min before quenching in binding-washing (B-W) buffer (10 mM Tris–HCl pH 7.5, 500 mM NaCl, 10 mM EDTA). Quenched reactions were incubated with ∼20 μl streptavidin coated beads for 1 h at 4°C to allow capture of the DNA templates and primase reaction products. Following capture, beads were washed three times with 1 ml volumes of B-W buffer before final suspension in 20 μl stop buffer (95% formamide solution with 0.25% bromophenol blue and xylene cyanol dyes). Samples were then boiled for 5 min and products detected by resolution on a 15% (v/v) polyacrylamide gel containing 7M urea and 1× TBE buffer run at 850 V for 2.5 h in 1× TBE. Reaction products were visualized using a FujiFilm FLA-5100 image reader.