Disposable plug mold

Sub-confluent cultures of U2OS were treated with vehicle alone (DMSO), camptothecin (CPT 1 μM), or Ru65 (50 μM) and were either non-irradiated or UV-A irradiated. Cells were harvested by trypsinization, and agarose plugs of 10⁶ cells were prepared in a disposable plug mold (Bio-Rad). Plugs were incubated in lysis buffer (100 mM EDTA, 1% (w/v) sodium lauryl sarcosyl, 0.2% (w/v) sodium deoxycholate, 1 mg ml^–1 proteinase K) at 37 °C for 72 h, and washed four times in 20 mM Tris–HCl pH 8.0, 50 mM EDTA before loading onto an agarose gel. Electrophoresis was performed for 23 h at 14 °C in 0.9% (w/v) Pulse Field Certified Agarose (Bio-Rad) containing Tris-borate/EDTA buffer according to the conditions described in47 (link) and adapted to the Bio-Rad CHEF DR III apparatus. The gel was finally stained with ethidium bromide (EtBr) and analyzed using an Alpha Innotech Imaging system.

Modified Pulsed Field Gel Electrophoresis (PFGE) Protocol for typing K.pneumoniae was used as described by Zakaria and Hassuna (2019) (link). Briefly, 24 K. pneumoniae isolates were recovered from −70°C stock culture and streaked on Mueller-Hinton agar (Oxoid, United States). All Culture plates were incubated at 37°C for confluent growth without exceeding a 24-h incubation. Plugs were made by adding an equal volume of molten 1% SeaKem Gold (SKG, Lonza, United States) agarose to the cell suspension, and the mixture was immediately dispensed into the wells of a disposable plug mold (Bio-Rad Laboratories, United States). Plugs were carefully transferred from the mold into new sterile 5 ml plain tubes containing 2 ml lysis buffer. Tubes were incubated overnight at 56°C in shaker incubator with vigorous agitation (170–180) rpm. Plugs were restricted with 50 U Xba1 enzyme (New England Biolabs, United States) and 2 μl BSA (10 mg/ml Thermo Fisher Scientific) and incubated for 16 h at 37°C. Digested slices were loaded into 1% seakem gold agarose with electrophoresis conditions: initial switch time 6.7 s and final switch time 35.6 s and 6 V for 19 h at 14°C. Lambda ladder (New England Biolabs) was used as a molecular size marker.

ES cells were transfected or treated as indicated and cells were harvested by trypsinization. Agarose plugs of 2.5 × 10⁵ cells were prepared in a disposable plug mold (Bio-Rad Laboratories). Plugs were then incubated in lysis buffer (100 mM EDTA, 1% (wt/vol) sodium lauroyl sarcosinate, 0.2% (wt/vol) sodium deoxycholate, and 1 mg ml⁻¹ proteinase K) at 37 °C for 72 h. Plugs were then washed four times in 20 mM Tris-HCl, pH 8.0 and 50 mM EDTA before loading onto an agarose gel. Electrophoresis was performed for 21 h at 14 °C in 0.9% (wt/vol) Pulse Field Certified Agarose (Bio-Rad Laboratories) containing Tris-borate/EDTA buffer in a pulse field gel electrophoresis (PFGE) apparatus (CHEF DR III; Bio-Rad Laboratories), according to the following protocol (block I: 9 h, 120° included angle, 5.5 V cm⁻¹, 30–18 s switch; block II: 6 h, 117° included angle, 4.5 V cm⁻¹, 18–9-s switch; block III: 6 h, 112° included angle, 4.0 V cm⁻¹, 9–5 s switch). The gel was then stained with ethidium bromide and analysed by the AlphaImager system (ProteinSimple). Relative DNA DSB) levels were assessed by comparing DSB signals for each treatment to the background levels observed in untreated conditions using ImageJ.