G.tigrina Hox gene DthoxC insertion into prokaryote E.coli / (by UNIamCloning)/2011/11/01

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Plasmid Isolation of Amplified GFP and ATF1 Genes

  • Today we isolated the plasmids containing our amplified genes from the competent cells. We began by making glycerol stocks of our cultures and storing them in the freezer just in case we should need them for whatever reason in the future. Then we isolated the plasmids by first spinning the cultures in the centrifuge, causing the cells to collect in sticky palettes at the bottom. Then we used Resuspension, Lysis, and Neutralization Solutions from the GeneJET Plasmid Miniprep Kit to lyse the cells and pipetted the supernatant into column tubes. The columns were "washed" using the Kit's Wash Solution, meaning all the junk that is not our desired DNA was "washed" through the filter and this flow-through was discarded. This was done twice, and then the purified DNA was eluted through the filter itself using the Elution Buffer from the Kit and collected in labeled 1.5mL tubes. The tubes were incubated for 2 min. and stored in the freezer.