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

Seattle Biomed Cloning Project

Steps leading up to transfection of Toxoplasma

Step 1: pGEM plasmids containing gene of interest are purified from E. coli bacteria using a Qiagen Plasmid Midi Kit.

Step 3: Transformation of E. coli with vector containing gene of interest, and selection of single colonies from LB-ampicillin plates.

Step 4: After single colonies are grown up in 2ml cultures, the vector containing our gene of interest is purified from the E. coli using a Qiagen Plasmid Miniprep Kit.

Step 2: Purified plasmids, along with the Toxoplasma expression vector, are cut with appropriate restriction enzymes, the gene of interest and expression vector are gel purified and then ligated together

Step 5: Purified vectors are cut with a restriction enzyme and analyzed by gel electrophoresis to confirm the presence of gene of interest, and sequenced.

Step 6: Vectors with gene of interest are digested with restriction enzyme to linearize, concentrated using Plasmid Midi Kit and ethanol precipitation, transfected into Toxoplasma, and gene products (proteins) are visualized by fluorescence microscopy.

For more information about my Summer Research Project at Seattle Biomed, please check out my blog: http://jmshaver.wordpress.com/. To find out what is happening in the Cleveland School of Life Sciences (SoLS), check out: http://sps.seattleschools.org/schools/cleveland/solshaps.html.