Our minipreps resulted in pellets of pAAV and f3-55nm DNA being successfully isolated from DH5-α bacterial cells. We successfully transformed pAAV, f3-55nm and fMCS plasmids into DH5-α bacterial cells as evident by the growth of bacterial colonies resistant to tetracycline. Our gels confirmed successful transformation of the f3-55nm-MCS plasmid.
Lawn growth observed on A1 and A2 f3-55nm plates. Individual colonies were picked from the edges.
The gel for the f3-55nm-MCS plasmid was not as clear. Possible reasons include not running the gel electrophoresis for long enough, and/or there could have been contamination with another DNA source. We discovered that the f3-55nm stock was contaminated with an unknown plasmid. This created challenges when isolating f3-55nm-MCS and produced unclear bands on the gels.
Gel electrophoresis of f3-55nm showing that the stock was contaminated with an unknown plasmid.
In the future, we plan to carry out more validation experiments. These include transduction of mammalian cells with our target peptide, as demonstrated by green fluorescent protein (GFP) expression and cell lysis. In addition, we will create our complete target peptide by digesting anti-CD22 with BglII and f3-55-MCS with SfiI, ligating these two sequences together, then transforming our phages.
Virtual digest of f3-55nm plasmid (1 = BamHI, 2 = SacII, 3 = BamHI + SacII)
Virtual digest of fMCS plasmid (1 = BamHI, 2 = SacII, 3 = BamHI + SacII)
Gel electrophoresis of digested pAAV and f3-55nm. Lane 1 = ladder, lane 3 = f3-55nm control, lane 4 = f3-55nm digest, lane 6 = pAAV control, lane 7 = pAAV digest. f3-55nm and pAAV digest were cut out and extracted from the gel prior to scanning.
- 1 band in the f3-55nm control lane.
- No bands in the f3-55nm digested lane.
- 3 bands in pAAV control lane.
- 1 band in pAAV digested lane.