Scott Casey ’15
Weeks 8 & 9:
University of Iowa Department of Biochemistry
July 15, 2013 | My eighth week in Maria Spies’ lab was spent restarting a cell line of HEK293T in order to transfect it to create FANCJ; hopefully with a higher concentration this time. The cell line was successfully restarted on Tuesday by switching to a different set of HEK293T stocks and was scaled up over the weekend in […]
Week 7:
University of Iowa Department of Biochemistry
July 3, 2013 | The fractions most likely to contain FANCJ were run on an SDS-Page gel which showed a very faint signal in Fraction 7 for FANCJ. Fraction 7 was analyzed on the Nanodrop spectrophotometer at 280 nm wavelength but there was no detectable concentration of FANCJ. Because the concentration of FANCJ in the fraction was so low […]
Week 6:
University of Iowa Department of Biochemistry
June 25, 2013 | A Western Blot was run on the eluate from the crude filtration and several fractions from the gel chromatography; unfortunately it showed that while the Eluate from the crude filtration did indeed have FANCJ in it, none of the fractions did. A second Western blot was run on the remaining fractions to determine if any […]
Week 5:
University of Iowa Department of Biochemistry
June 18, 2013 | The purified plasmid containing the FLAG-tagged FANCJ was run on an agarose gel to confirm the proper vector and insert were present. The Gel showed that they were. Following the confirmation of FANCJ in the purified plasmid, the cultures of mammalian cells, HEK293T, were transfected and left to incubate overnight. The protein from the transfected […]
Week 4:
University of Iowa Department of Biochemistry
June 11, 2013 | A bulk fluorometry was run on the elution from the crude filtration of last week to determine the elution’s concentration of FANCJ. This assay is possible because of the structure of FANCJ which contains an iron-sulfur cluster. This cluster quenches the fluorescence of cytochrome 3 and 5 when in close proximity, such as when it […]
Week 3:
University of Iowa Department of Biochemistry
June 4, 2013 | The frozen cells from the previous week were thawed and put through a process called crude filtration. The cells were lysed and the lysate was then filtered before being mixed with beads coated in an anti-FLAG protein that will bind to the flag tag on our FANCJ and hold it to the beads. The bead […]
Week 2:
University of Iowa Department of Biochemistry
May 28, 2013 | The first week of my internship at Maria Spies single molecule biochemistry lab was spent familiarizing myself with the lab and general protocols and beginning the work of creating the protein stocks that I need for my investigation of FANCJ and it’s binding and motility properties. This first week was both very busy and very […]