Ez 10 spin column bacterial genomic dna miniprep kit

Genomic DNA from each isolate was extracted using EZ-10 Spin Column Bacterial Genomic DNA Miniprep Kit (Biobasic, Markham, ON, Canada). Presence and integrity of DNA was checked on agarose gel and its quantity was estimated using nanophotometer (Implen, Munich, Germany). Concentration of DNA was normalized to 20 ng∙µL⁻¹.
The presence or absence of bla_KPC gene was confirmed by PCR using specific primers [76 (link)] which amplify complete sequence of bla_KPC gene family. Thirty µL of PCR mixture contained Dream Taq Green buffer 1×, 2 mM of DNTP mix, 1 U of DreamTaq polymerase (All chemicals from Thermo, Waltham, MA, USA), 0.5 µM of primer Uni-KPC-F (5′-ATGTCACTGTATCGCCGTCT-3′) 0.5 µM of primer Uni-KPC-F (5′-TTACTGCCCGTTGACGCCC-3′), and 1 µL of extracted DNA.
The amplification was performed using MJ Mini Thermal Cycler (Biorad, Hercules, CA, USA) with the following settings: 95 °C for 3 min for initial denaturation, followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, and a final extension step at 72 °C for 5 min.
Presence of PCR product was detected using agarose gel electrophoresis (1.5% gel, 1 X TAE buffer, 5 µL DNA loaded). Migration speed was compared to 100 bp plus ladder (Thermo, Waltham, MA, USA) after visualization with ethidium bromide. Isolate was scored as positive for bla_KPC gene if PCR product of approximately 900 bp was detected.

Template DNA was extracted using an EZ-10 spin column bacterial genomic DNA miniprep kit (Bio Basic Inc., Markham, ON, Canada). Genes encoding AMEs, including aac(6')-Ie-aph(2''), aph(2'')-Ib, aph(2'')-Id, aph(3')-IIIa, ant(3'')-I, ant(4')-Ia, and ant(6')-Ia, along with the virulence genes ace, asa1, cylA, efaA, esp, gelE, and hyl, were examined by PCR. Detection was performed in a final volume of 20 μL, containing 400 nM of each primer (primer sequences are provided in Table 1), 10 µL of premix Taq polymerase (Takara, Otsu, Japan) containing MgCl₂, dNTPs, and reaction buffer, 1 µL of DNA template, and dd H₂O to 20 µL. The PCR conditions consisted of a pre-denaturation step at 94 °C for 4 min, followed by 35 cycles of 94 °C for 40 s, 55 °C for 40 s, and 72 °C for 45 s. A final extension step was performed at 72 °C for 5 min.
The amplified PCR products were analyzed on 1% (w/v) agarose gels. DNA bands were visualized by staining with ethidium bromide and photographed under UV illumination.