1. Ana Velázquez and Michael Rubin RISE Program Department of Biology University of Puerto Rico at Cayey
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10. Add competent cells to ligation reaction. Ice for 20min Heat shock Put on ice Add SOC medium to the tubes: -Transformed cells (950µL) Incubate Add -900µL -50µL to plates Incubate plates overnight Take one colony with micropipette tip from 50µL plate and put it in LB stock with 50µg/mL of ampicillin Cultivate Transfer LB broth to micro centrifuge tube. Now we have cells with the desired plasmid Centrifuge
11. -Add resuspension solution to the cells (200µL) -Centrifugate and remove flow-through -Add matrix solution (200µL) -Remove supernatant -Discard the precipitate -Add neutralization solution (250µL) -Centrifugate -Add lysis solution to the cells (250µL). -Store column with purified plasmid -Add wash buffer (200µL) to the plasmid. -Centrifugate for 30sec