Seattle Biomed Cloning Project Jeff Shaver, Biomedical Teacher (CTE, PLTW)

Cleveland High School

Day 1: Run Polymerase Chain Reaction (PCR) using cDNA library and primers specific to gene of interest.

Day 3: Ligate DNA fragment to pGEM plasmid and transform E. coli with plasmid containing gene of interest.

Day 4: Select white colonies and grow up 2ml cultures.

Day 2: Run PCR reactions out on gel, excise DNA band of interest, and purify the DNA from the gel.

Day 5: Isolate plasmids from bacterial cultures, cut plasmids with EcoR1 restriction enzyme, and analyze by gel electrophoresis. Then, sequence plasmid samples that have desired fragment size on gel.

Day 6 or 7: Obtain sequence data, and determine if plasmid samples contain gene of interest. Then, proceed with the multi-step, -day process of ligating (inserting) gene into vector for transfection of Toxoplasma.

Thanks to Seattle Biomed, particularly the Parsons lab, for providing me the opportunity to be a visiting scientist this summer (2011). Available at http://prezi.com/7cl67mui-ley/seattle-biomed-cloning-project-summer-2011