Journal: Nucleic Acids Research
Article Title: Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed
Figure Lengend Snippet: Quantitative AGE assay development. ( A ) Ethidium bromide versus Sybr-Gold ‘Staining’. Aliquots of 200 ng of four plasmid batches known to contain differing levels of recombinant plasmid were separated by AGE and then stained with 1× TBE containing either ethidium bromide (50 ng/ml) (left gel) or 1× Sybr-Gold (right gel) for 30 min prior to gel-image capture by Polaroid camera. Arrows indicate the position of low-level recombinants only clearly visible with Sybr-Gold staining. ( B ) Image analysis of a 1× Sybr-Gold stained gel using manufacture's recommended staining times. Image capture by ProXpress. For this analysis, 1, 2 and 5 μl volumes of MassRuler High Range DNA Ladder (MBI-Fermentas, Lithuania) were loaded in lanes 1, 2 and 3, respectively. Left panel, gel image with loads corresponding to DNA quantities (per band) ranging from 1.6 to 10 ng (lane 1), 3.2 to 20 ng (lane 2) and 8 to 50 ng (lane 3). Middle panel, schematic ImageQuant (Molecular Dynamics, SunnyVale, CA) generated electropherogram traces of each lane. Right panel, scatter-plot with trend-line representation of area-under-curve signal ( y -axis) against quantity (in nanograms) of DNA ( x -axis) of the combined 1.6 ng through to 50 ng results. This plot highlights the significant signal ‘plateau’ that develops above 50 ng loads ( C ) Extending assay linear range by increasing Sybr-Gold staining times. Triplicate 5 μl (lanes 1, 3 and 5) and 30 μl (lanes 2, 4 and 6) MassRuler DNA ladder loads were separated by AGE. Subsequently, the gel was then dissected longitudinally into three parts and stained for 20 min (lanes 1 and 2), 2 h (lanes 3 and 4) or 24 h (lanes 5 and 6). After such staining regimes, excess stain was removed by copious washing with water. Image analysis by ProXpress and scatter plot data representation was then undertaken as described above in (B). The 5 and 30 μl loads generate a collective DNA species quantitative range of between 8 and 300 ng. The results of analysis reveal the R 2 values obtained for the 20 min, 2 h and 24 h staining regimes are 0.910, 0.990 and 0.999, respectively.
Article Snippet: The 1× TBE running buffer also contained 5.0 μg/ml chloroquine, and electrophoresis was performed at 50 V for 48 h. Staining was carried out by soaking in 1× TBE containing 1× Sybr-Gold (Molecular Probes) for 24 h in the dark.
Techniques: Staining, Plasmid Preparation, Recombinant, Generated