The use of Gibson Assembly for DNA cloning

Abstract: This thesis report revolved around the cloning process of plasmids. Attempts of cloning the red fluorescent protein mCherry, and the green fluorescent protein EGFP from various plasmids, into other plasmids containing different cell-junction/cytoskeleton plasmids were made. These plasmids were first amplified using PCR, and then cloned using Gibson-Assembly, and then transfected into live HEK293T or MDCK-II cells. After the transfection, the cells were examined in a microscope. The results showed no signal or localization for the cloned plasmids in their respective corresponding channel, 561 nm for the red fluorescent protein mCherry or 488 nm for the green fluorescent protein EGFP. The step that went wrong was the PCR step in the cloning process, since the backbone vector was not successfully amplified. The reasons for this was either that the backbone vector was too long, the primers regions were to rich with Guanine and Cytoseine, or the primers being too long.