Chef dr 3 variable angle system

90% confluent U-2 OS cells grown on 6 cm dishes were treated as indicated and then trypsinized. 1 × 10⁶ cells per condition were resuspended into 25 μl of PBS, then embedded into 60 μl of molten 0.9% Low Melting Point Agarose (Invitrogen) in 0.5× TAE Buffer (20 mM Tris pH 8.5, 10 mM acetic acid, 0.5 mM EDTA). The mixture was cast into a mold on ice to form a gel plug of volume 80 μl. Each plug was then digested with agitation at 32°C in 0.5 ml of PFGE Lysis Buffer (100 mM EDTA, pH 8.0, 1% N-laurylsarcosine, 0.2% sodium deoxycholate, 1 mg/ml Proteinase K) for 48 h. The treated plugs were then washed three times 15 min each in TE Buffer (20 mM Tris–HCl pH 8.0, 50 mM EDTA) with gentle agitation at room temperature, then cut in half. The half-plugs were electrophoresed at 14°C, in duplicate, in a 0.9% Certified Megabase Agarose (Bio-RAD)/0.5× TAE gel containing 0.25 μg/ml ethidium bromide via the CHEF-DR III Variable Angle System (Bio-RAD). The running parameters were as follows: Block 1 (9 h, 120° included angle, 5.5 V/cm, 30–18 s switch time); Block 2 (6 h, 117° included angle, 4.5 V/cm, 18–9 s switch time); Block 3 (6 h, 112° included angle, 4.0 V/cm, 9–5 s switch time). Resolved gels were visualized on a Typhoon 9400 Variable Mode Imager (GE Healthcare), then quantified with ImageQuant 5.2 (GE Healthcare).

PFGE was performed as described in [66 (link)], and the CHEF-DR® III variable angle system was used for gel electrophoresis (Bio-Rad). 150 ml 1% PFGE certified agarose (Biozym Gold Agarose) prepared in 0.5 × TBE was used for gel casting. The gels were run in 0.5 × TBE for 24 h under the following conditions: 6 V/cm, 120 degree included angle, 8–50 sec switch time ramp, 14°C.

MLST and PFGE were used to determine the genetic relatedness among the 45 CRKP isolates. PCR and sequencing for MLST were carried out for seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) for K pneumoniae and the sequences were compared in the MLST database, so that the allelic numbers and sequence types (STs) could be determined.³³ The allelic profiles and STs were assigned using an online database (https://pubmlst.org/). For PFGE, bacterial DNA was cleaved with XbaI endonuclease (Roche, Penzberg, Germany), and the XbaI‐digested genomic DNA was subjected to PFGE using a CHEF‐DR^® III Variable Angle System (Bio‐Rad, USA).³⁴ The PFGE patterns were compared using BioNumerics software (Applied Maths, Kortrijk, Belgium) with dice correlation for band matching at a 1.5% position tolerance and the unweighted pair group method with an arithmetic average (UPGMA). Clusters were defined as DNA patterns sharing >80% similarity.