Dna gel loading dye

Genotyping was performed by lysing ear cuts in 50 mM Tris-HCl, 0.45% Igepal v/v, 0.33 mg/mL Proteinase K at 55 °C overnight under constant agitation. The next day, samples were centrifuged at 16.000 g for 1 min. Supernatant was incubated at 95 °C for 10 min to inactivate Proteinase K. PCR was performed using SYBR green (Roche, #04913914001). PCR cycles consisted of denaturation at 94 °C for 30 sec, annealing at 56 °C for 30 sec and extension at 72 °C for 2 min for 35 cycles. PCR products were run combined with DNA Gel Loading Dye (Thermo Scientific, #R0611) on a 1% agarose gel (89 mM Tris-HCl, 89 boric acid, 2 mM EDTA) mixed with 0.01% v/v SYBR® Safe DNA Gel Stain (Invitrogen, #S33102) to visualise DNA.

According to the molecular weight of the separated DNA, a gel with the corresponding concentration was prepared. The solvent for the gel was 0.5×Tris-borate-EDTA (TBE), and the solute was agarose powder. GelRed (Solarbio, China) was added to the gel at a ratio of 1:10,000. DNA samples (10 ~ 20 μL) that were previously mixed with DNA gel loading dye (Thermo Scientific, US) were added to the gel, and electrophoresis was performed. The strips were visualized using a chemiluminescence imager (Bio-Rad, US) and analyzed using Image Lab Software (Bio-Rad, US).

Heparin sodium salt (H4784-1G) and CaCl₂ were from Sigma-Aldrich. DNA Gel Loading Dye (R0611) and Lipofectamine 3000 were from ThermoFisher, molecular weight markers Sky High (10 and 500 bp) were from BioLabMix, Russia. MTT (3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide was from Sigma-Aldrich. Plasmid pEGFP-C1 (GenBank Accession U55763; cat 6084-1) was purchased from BD Biosciences Clontec, monoclonal anti-RL2 mouse IgG (clone F14; Biosan, Russia).
The recombinant analogue of lactaptin RL2 was obtained from E. coli and purified as described previously (Semenov et al., 2010 (link)). The 98% purity of the isolated protein was confirmed by RP-HPLC chromatography on C5 reverse phase column (Discovery BIO Wide Pore C5; Sigma) in water (0.5% TFA)-acetonitil solvent system using HPLC Station (Bio-RAD Laboratories) as well as by RP-HPLC on C18 (ProntosSIL) using Milichrom A-02 station (EcoNova, Russia) (Supplementary Figure 3A) and by SDS-PAAG electrophoresis under reduction conditions (Supplementary Figure 3B).

The Acinetobacter species PCR identification protocol was modified from previous studies (19 (link)). Briefly, a clinical isolate from each patient was separately grown on LB agar at 37°C overnight. Genomic DNA was extracted by using boiling method (35 (link)); approximately three to five isolate colonies were resuspended in sterile deionized water and then boiled at 95°C for 10 min. After centrifugation at 12,000 × g for 10 min, supernatants were collected and estimated for DNA concentration by measuring the absorbance at 260 nm. PCR was performed according to the GoTaq Flexi DNA polymerase (Promega) manufacturers' instructions with GeneAmp PCR System 2700 (Applied Biosystems) thermocycle settings as follows: 94°C for 5 min, followed by 45 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 10 min. The primers used in this study and their interpretation are shown in Table S1.
The PCR amplicons were separated by electrophoresis (100 V, 80 min) in 1.5% agarose gel in 40 mM Tris, 20 mM boric acid, and 1 mM EDTA (TBE) buffer pH 8.3 containing DNA Gel Loading Dye (Thermo Fisher Scientific). The gels were visualized and captured using a gel image analysis system (UVitec, Cambridge, United Kingdom). The multiplex PCR results were validated for the first 18 isolates using 16S rRNA sequencing (Macrogen, Inc., South Korea) (Fig. S1).

PCR was carried out with AmpliTaq Gold^® DNA polymerase with Buffer II and MgCl₂ (Thermo Fisher Scientific). Taq polymerase and Buffer II were added as per the manufacturers instructions; additionally, one PCR volume contained 0.5 mM MgCl₂, 5% dimethyl sulfoxide (DMSO), 1 M N,N,N-trimethylglycine (betaine), 0.1 μM dNTPs, 0.5 μM forward primer, 0.5 μM reverse primer, and 1.5 μl of sample DNA (1 μg/μl). The PCR was performed on a MiniOpticon PCR System (BioRad Laboratories, Solna, Sweden) or a 2720 Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific) with the following protocol: one cycle of 95°C for 3 min; 39 cycles of 95°C for 30 sec; 56, 60, 65 or 70°C for 30 sec; 72°C for 1 min; and one cycle of 72°C for 7 min. PCR products and the GeneRuler 100 bp or 1 kb DNA ladder (Thermo Fisher Scientific) were diluted in DNA Gel Loading Dye (6X; Thermo Fisher Scientific) and separated by electrophoresis on 1% or 2.5% agarose gels, prepared in 40 mM Tris, 20 mM acetate and 1 mM EDTA (TAE buffer) with 1‱ GelRed fluorescent DNA stain (Biotium, Hayward, CA, USA). Gels were scanned using a Gel Do EZ imaging system (BioRad Laboratories) or an Odyssey Fc Imaging System (LI-COR Biosciences, Cambridge, United Kingdom) and images were collected using the ImageLab Software (BioRad Laboratories) or Image Studio^™ Software (LI-COR Biosciences).