Doc print 2

The nuclease activity of the compounds was determined using plasmid pRS325II DNA, which was a gift from Steven Haase (Addgene plasmid #35467, 6835 base pairs) [71 (link)]. The plasmid was amplified in Escherichia coli by transforming One Shot^® TOP10 chemically competent E. coli (Invitrogen, Thermo Fisher Scientific). The plasmid was isolated from positive colonies using a PureLink™ HiPure Plasmid Miniprep Kit (Invitrogen, Thermo Fisher Scientific). Plasmid (200 ng/sample) was exposed to complexes (1) and (2) in the presence of 1 mM hydrogen peroxide (to exploit the Cu(II/III) redox couple) or 1 mM ascorbic acid (to exploit the Cu(I/II) redox couple) [46 (link)]. The DNA cleavage experiments were done in a 9/1 (v/v) ratio of 50 mM Tris-HCl, pH 8, and DMSO. The samples were incubated for 1 h at 37 °C before a bromophenol blue/xylene cyanol-based loading dye (Roth, Germany) was added. The samples were loaded onto a 1% (w/v) agarose gel containing 1 μg/mL EtBr in 1 × TBE (Tris–boric acid–EDTA) buffer. Electrophoresis was performed at 50 V for 60 min in 1 × TBE buffer. The images of the fluorescent ethidium bromide-stained gels were captured using a gel documentation system (Doc-Print II, VilberLourmat, France). The cleavage experiments were done three times, with similar results. One representative gel is shown.

The previously selected set of 14 microsatellite loci ORPM193, ORPM202, ORPM206, ORPM220, ORPM296 [11 (link)], WPMS14, WPMS15, WPMS16, WPMS17, WPMS18, WPMS19, WPMS20, WPMS21, and WPMS22 [12 (link)] was employed for DNA identification [7 (link), 8 (link)] of “Meshabash” clone ramets from putatively growing together in the same plot other aspen genotypes/clones.
Reagents used for PCRs, laboratory equipment, regimes of DNA amplification, fragment analysis, and gel documentation were performed as described earlier [7 (link), 8 (link)]. PCR products were analyzed by electrophoresis in vertical blocks of 6% polyacrylamide gel in the tris-EDTA-borate buffer system. After electrophoresis, the gels were stained in a solution of ethidium bromide and visualized under UV light and their graphic images were captured using the Doc-Print II (Vilber Lourmat) gel documentation system. The size of amplified fragments was estimated with the help of the Photo-Capt software.

The obtained electrophoretic patterns of H1 histones were recorded with the gel imaging system Doc-Print II (Vilber Lourmat) and processed by the software ImageJ 1.44c (www.rsbweb.nih.gov/ij). A raw integrated density, which indicate a sum of the values of the pixels in the selected gel area, was measured to evaluate an abundance of histone H1.2 phenotypes protein spots. The measurements were repeated for the seven preparations (n = 7) of each histone H1.2 phenotype.