1 kb dna ladder

DNA was extracted from pure bacterial cultures by boiling for 15 min at 95 °C. PCR reactions were then prepared as follows: 3 µL of DNA, 0.5 µL of each of the forward and reverse specific primers, 4 µL of master mix (5× FIREPol^® Master Mix Ready to Load, Solis BioDyne, Tartu, Estonia), and 12 µL of DNase free water. The identity of the E. coli isolates was further confirmed by targeting a species-specific 16S rRNA gene fragment. The PCR analysis also included screening for mcr-1 and other mcr genes (mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7, mcr-8) (Table 1). Reactions without DNA were used as a negative control, while DNA from a previously confirmed mcr-1-positive E. coli was used as a positive control [35 (link)]. The amplified products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide (BioRad, Hercules, CA, USA) and visualized using a gel imaging system (BioRad, Hercules, CA, USA). 1 Kb DNA ladder (Solis BioDyne, Tartu, Estonia) was also loaded in the gel to determine the size of the amplicons.
Using PCR analysis, the E. coli isolates were also screened for other genes, including bla_TEM-1, bla_CTX-M, bla_SHV-1, bla_NDM-1, bla_OXA-48, bla_IMP, bla_KPC, int1, Class 1 Integron gene, Class 2 Integron gene, tetA, tetB, tetC, tetD, tetE, tetG, and strA (Table 1).

All chemicals and solvents used were of analytical grade. Copper chloride, thymus DNA (CT-DNA, calf thymus) and the kaempferol flavonoid were purchased from Merck (Darmstadt, Germany), and bromphenol blue from AppliChem GmbH (Darmstadt, Germany). Dimethyl sulfoxide and glycerol were obtained from Merck (Darmstadt, Germany), hydrogen peroxide from Mikrochem (Pezinok, Slovakia), and tryptone and agar from Merck (Darmstadt, Germany). ABTS ((2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) was obtained from Merck (Darmstadt, Germany). The 1 kb DNA ladder was from Solis BioDyne (Tartu, Estonia), ethidium bromide (EtBr) was from Serva (Heidelberg, Germany), EcoR1 restriction was from Thermoscientific (Rockford IL, USA), and the Isolate II Plasmid Kit from Bioline (Memphis TN, USA). Tris-NaCl buffer (5 mM TRIS, 50 mM NaCl, pH 7.2) was from Aniara Diagnostica (West Chester OH, USA).