Monday, April 10, 2006

About the Project

I'm a high school senior, attending MIT in the fall. My past AP biology teacher offered me a class period with him this year during which I could use the school's equipment to do any projects I desired. I found a protocol outline online from Davidson College, and updated it from its most recent 2000 revision. Ultimately I modified about 95% of it, using newer systems and kits.

In brief outline, the project is as follows:
  1. Purify yeast genomic DNA from a saturated culture.
  2. Amplify the target gene, Isocitrate Dehydrogenase, NADP+ specific (IDP2).
  3. Verify on gel.
  4. Clone gene using Novagen Perfectly Blunt cloning system. This is a blunt ligation with pETBlue into NovaBlue singles for plasmid screening.
  5. Screen colonies. If my PCR turns out pretty clean, then I'll just use the extra plasmid preps that I have and do a restriction digestion. If it's dirty, I'll buy the Epicentre restriction screen.
  6. Plasmid prep from screened colonies.
  7. Transform Tuner (DE3)pLacI. Induce expression.
  8. Affinity purify the protein using Sigma gravity flow column.
  9. Run SDS-PAGE to determine size of protein.
My budget is approximately $750, of which I've spent $745 on everything but a possible functionality assay at the end and any incidentals. (I might have an additional $70 from a photoshoot I just did.)

I'm working under the auspices of Mr. Rene Gillibert, biology teacher and science department head at Campolindo; and the assistance of Ms. Marietta Dunaway, biotechnology teacher at Campolindo. I am sincerely greatful for all their help! Special thanks as well to Vicky Kim at Novagen for her donations of markers and induction media and discount; to the tech people at Epicentre and sales people for offering a discount; to the sales people at USB for offering a discount; and to Janet Long at John Muir Regional Medical Center for allowing me to store supplies in her -70 freezer.


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