The document discusses a study on metalloproteases (MP), enzymes that shape the extracellular matrix. The researchers aim to control irregularities in MP activity by inhibiting the MP gene. They plan to obtain and sequence the MP gene from the Bornean orangutan (Pongo pygmaeus) to gain information on a global health issue. Their goals are to purify and sequence the MP gene, transform E. coli with the gene for expression, and perform protease detection assays.

1. Ana Velázquez and Michael Rubin RISE Program Department of Biology University of Puerto Rico at Cayey

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

12. Marker Pongo pygmaeus PCR 150bp 200bp 300bp 400bp 500bp 600bp 700bp 1,200bp 1,400bp Work by: Angiemar Maldonado

13. Marker Pongo pygmaeus cloning Marker 1,200bp Work by: Angiemar Maldonado Vector

16. Ana Velázquez and Michael Rubin RISE Program Department of Biology University of Puerto Rico at Cayey