Gel loading dye blue

To assess Hduc- or EcolCdtB nuclease activity in vitro, 250 ng or 125 ng of the supercoiled plasmid pRSET-A (Invitrogen) resuspended in water was incubated at 37°C with 9 ng to 1 μg of the CdtB subunit, for 30 min to 7 h, in digestion buffer (20 mM TrisHCl pH 7.5, 50 mM NaCl, 5 mM CaCl2, 5 mM MgCl2, 50 μg/mL BSA), containing 10 mM of divalent cation as recommended previously [18 (link)]. The reaction was stopped by adding 10 mM of EDTA (final concentration) and Gel Loading Dye blue (1x final concentration, New England Biolabs). The digestion products were analysed on 1% agarose gel (migration 25 min at 75 V), stained by ethidium bromide. Plasmid was incubated at 37°C in absence of CdtB subunit or in presence of HducCdtC for negative control, or in presence of recombinant bovine DNase I (2000 u/mL, New England Biolabs) for positive control. The band intensities of supercoiled, relaxed and linear plasmids were quantified with ImageJ software.

The PR genotype was identified from retrospectively collected blood samples stored at −80°C until analysis. Genomic DNA was extracted from 200 μL of blood using the QIAamp DNA Blood Mini Kit (QIAgen, Hilden, Germany) and an automated procedure (QIAcube, QIAgen, Hilden, Germany). PCR was performed with MyTaq^TM DNA Polymerase (Bioline, Meridian Life Science, Memphis, TN, United States) together with the following primers (20 μM) used to detect intron G and identify the PR genotype: forward primer 5′-GCCTCTAAAATGAAAGGCAGAAAG-3′ and reverse primer 5′-GTATTTTCTTGCTAAATGTCTG-3′ (Agoulnik et al., 2004 (link)). The thermal profile was 95°C for 1 min followed by 35 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 10 s. Electrophoresis was conducted with 10 μL of PCR products mixed with 1.6 μL of (6×) Gel Loading Dye, Blue (New England BioLabs) on a 2% agarose gel with 5 μL of ethidium bromide in a volume of 150 mL for 50 min at a constant voltage of 90 volts. The Tracklt 100-bp DNA ladder (Invitrogen, Burlington, ON, Canada) was used to identify the molecular weight of the bands in the agarose gel. The bands were visualized using the Molecular Imager Gel Doc XR System (BioRad, Hercules, CA, United States).