Transilluminator

A. baumannii (ATCC BAA‐1709) was cultured in MHB medium overnight (100 rpm, 37 °C). The bacterial suspension (50 mL) was then centrifuged and the pellet was lysed by G2 buffer. The genomic DNA was extracted using an EZ1 DNA Tissue Kit (48) in the EZ1 machine (QIAGEN) following the EZ1 DNA Tissue protocols. The gene binding ability of pEt_20 was investigated by agarose gel electrophoresis. DNA extracted from A. baumannii BAA‐1709 was mixed with pEt_20 at various polymer to DNA mass ratios (1–20). Briefly, 9.5 µL of the pEt_20/gene complex solution containing 300 ng of gene and corresponding polymer at their respective mass ratios was mixed with 0.5 µL of 5×  DNA loading dye. The mixture (7 µL) was loaded into individual wells of 1% agarose gel containing SYBR Safe DNA Gel Stain (Thermofisher Scientific, USA) at a ratio of 1:10 000 1× TAE buffer. The same amount of naked DNA was used as the control. The gel was run at 120 mV for 20 min in 1× TAE buffer. Following completion of the assay, the gel was imaged using a gel imaging system fitted with a transilluminator (Bio‐rad, U.S.A.).

DNA was extracted from SKBR3 treated cells as previously described [73 (link)], with some modifications. The cells were detached from the flasks with trypsin and suspended in 0.5 ml of TE buffer (100 mM Tris-HCl, 100 mM EDTA pH 8). 50 µl of 10% SDS and 25 µl of 1 mg/ml Proteinase K were added to the cell suspension and incubated for 30 minutes at 55°C. Nucleic acids were extracted using phenol-chloroform, precipitated with 1/10V 3M Na-acetate and 2V absolute ethanol and resuspended in 100 µl TE buffer. The same quantity of extracted DNA was loaded on a 1% agarose gel prepared in 0.5X TE buffer containing gel red. Gel was visualized under UV light using a Bio-Rad Trans illuminator and photographed by using a Polaroid camera.
Apoptosis of SKBR3 cells after treatment with IC₅₀ of AgNPs-EPS for 24 h was analyzed by flow cytometry essentially as described by Riccardi [74 (link)]. Briefly cells were fixed in 70% ethanol, washed in PBS and resuspended in DNA extraction buffer (200 mM Na2HPO4, 0.1% Triton X-100). After staining with Propidium Iodide (1µg/mL) for 30 min, fluorescence intensity was acquired in the FL2 channel by flow cytometry on a FACSCalibur flow cytometer (BD, New Jersey, USA). Data acquisition was performed with CellQuest Pro (BD) software, and analyzed with WinList software (Verity Software House, Topsham, USA).

The PCR products were analyzed via electrophoresis using a 1 % agarose gel in TBE
buffer (0.089 M Tris-Borate and 0.002 M EDTA) at a constant voltage of 100 V.
The gels were visualized under ultraviolet light using a transilluminator (Bio
Rad) and photographed with a photo-documentation system (Photocap, Vilber
Lourmat). The amplicons for each gene were purified using the SV Total DNA
Isolation System (Promega) and the DNA was sequenced by the method of Sanger et
al. (1997). The nucleotide sequences were analyzed using the BLAST program
(http://www.ncbi.nlm.nih.gov/).
Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction
(ERIC-PCR)
To assess the clonal relationship between the isolates, ERIC-PCR was performed as
described by Duan et al. 2009²⁹ and Cabral et al. 2012¹⁴ (Table 1). The DARWIN 6.0
software was used to generate the dendrogram.

One microgram of DNA from BACs F2D9, F2G3, F2G18, F2E13, and F2I6 were tested for the presence or absence of the restriction site (AvaI) at position 4133 of the reference rDNA. The region harboring the restriction site was amplified by PCR (primer sequences: AVAI Fw 5′-CGCATCAGCAAAGGATGATGG-3′, AVAI Rv 5′-ACCTTGGGATGGGTCGG-3′). The final amplicons were digested with the enzyme AvaI. Seventeen microliters of the PCR reaction were mixed with 2 µl of CutSmart Buffer and 1 µl of AvaI (NEB) for a final volume of 20 µl. The reaction was incubated at 37 °C overnight. The samples were loaded on an agarose gel (1 % w/v + EtBr) and visualized on a Biorad transilluminator.

DNA cleavage experiments were carried out according to the previously described procedure [39 ]. Briefly, the solution of compounds in DMF (1 mg/mL) was prepared and these test samples (1 µg) were added to the 500 ng of Calf thymus-DNA (CT-DNA) in TE buffer and incubated for 2 h at 37 °C. Agarose gel electrophoresis was performed after loading the samples on to the gel in TAE buffer system at 50 V for 2 h. At the end of electrophoresis, the gel was carefully stained with EtBr (Ethedium bromide) solution (10 µg/mL) for 10–15 min and visualized under UV light using a Bio-Rad Trans illuminator and the images were captured.