We evaluated the ability of Man-PEI to complex with a plasmid-based on a gel retardation assay, using agarose gel electrophoresis. Briefly, we complexed the Man-PEI at different nitrogen to phosphate (N/P; indicates the ratio between the number of nitrogen atoms present in the Man-PEI to the phosphate atoms in the nucleic acid) ratios and run the samples in a 1% agarose gel with 0.1 μg/mL ethidium bromide, as previously described [29 (link)]. The nucleic acid’s signal was visualized under a Chemidoc Touch Imaging (Biorad, Hercules, CA, USA).
To evaluate the ability of Man-PEI to protect nucleic acid degradation in the presence of DNAses, we complexed the pGL-3 plasmid with Man-PEI at different N/P ratios and incubated the samples in the presence of DNase I (2U of DNase/600 ng of plasmid for 30 min at 37 ºC; naked plasmid with DNAses was used as positive control). Following the deactivation of the DNAses using EDTA, the nucleic acids were released from the complexes using 8% polyacrylic acid (PAA) and run in the agarose gel, as described above.
To evaluate the ability of Man-PEI to protect nucleic acid degradation in the presence of DNAses, we complexed the pGL-3 plasmid with Man-PEI at different N/P ratios and incubated the samples in the presence of DNase I (2U of DNase/600 ng of plasmid for 30 min at 37 ºC; naked plasmid with DNAses was used as positive control). Following the deactivation of the DNAses using EDTA, the nucleic acids were released from the complexes using 8% polyacrylic acid (PAA) and run in the agarose gel, as described above.
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