Dig high prime labeling mix

Plasmids were purified by Gene Jet plasmid Miniprep kit (Thermo Scientific, Lithuania) and were subjected to transformation by heat shock method using E.coli JM107 as recipient. Transformants were selected on LB agar with 100μg/ml of ampicillin and 0.25μg/ml of imipenem. Southern blotting was performed on agarose gel by in-gel hybridization with the bla_KPC and bla_NDM probe labeled with digoxigenin DIG-high prime labeling mix (Roche, Germany) detection Kit. Plasmid DNA from transformants was separated by pulsed field gel electrophoresis (PFGE) (CHEF DR-III System, Bio-Rad; USA) and was transferred to nylon membrane (Hybond N, Amersham, UK), hybridized with prepared probes. Detection was performed using NBT color detection Kit (Roche, Germany).

Plasmid p24835-NDM5 was purified by a commercial kit (Qiagen, USA) and used to transform electrocompetent recipient K. pneumoniae BD2411. Briefly, 10 μl of plasmid DNA (50 ng) was mixed into 100 μl of electrocompetent K.pneumoniae BD2411 recipient in a microcentrifuge tube. After placing the mixture on ice for 30 min, the tube was placed into a 42 °C water bath for 45 s and then put back on ice for 2 min. Luria-Bertani broth was added into the tube and the mixture grew in 37 °C shaking incubator for 45 min. Transformants were selected on LB agar with 0.5 μg/ml of imipenem. Southern blotting was performed on agarose gel by in-gel hybridization with the bla_NDM probe labeled with digoxigenin DIG-high prime labeling mix (Roche, Germany) detection Kit. Plasmid DNA from transformant was separated by S1-PFGE and was transferred to nylon membrane (Hybond N, Amersham, UK), hybridized with prepared probes.
Stability of p24835-NDM5 was conducted as described previously [10 (link)]. Plasmid stability was estimated by propagating monocultures for ∼100 generations as well as of the transformant in 1:1000 dilutions without any antibiotic pressure. One hundred colonies were selected on LB agar with 0.5 μg/ml imipenem. Plasmid presence was checked by colony PCR targeting bla_NDM-5 and repB or repA. The stability test was performed three times, and the average values are presented.

To validate our study, Southern blotting was performed on agarose gel by in-gel hybridization [26 ] with the bla_CTX-M-15 probe labelled with Dig High Prime Labeling Mix (Roche, Germany) detection Kit. The dig-oxigenin-labeled bla_CTX-M-15 specific probe was prepared using primers (CTX-M-15 Forward 5’ CGCTTTGCGATGTGCAG3’ and Reverse 5’ ACCGCGATATCGTTGGT 3’) that amplify a 550 bp region of the bla_CTX-M. Separated plasmid DNA on agarose gel was transferred to nylon membrane (Hybond N, Amersham, UK) and then hybridised with prepared bla_CTX-M specific probe. Detection was performed by using an NBT color detection kit (Roche, Germany).