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 binds to DNA tagged with cytrochrome 5.  The DNA construct used for this assay is forked DNA that has a cytochrome 5 tag on one of the single strands of DNA that make up the fork.  When FANCJ binds to the single strand it quenches the fluorescent signal from the tag which can be detected by the fluorometer.  When the quenching first levels off the FANCJ and DNA are present in the solution at a 1:1 ratio which can then be used to back calculate the concentration of the elution.  The downside to this assay is that it requires a somewhat large amount of protein in a relatively high concentration in order to be effective which is where I ran into problems because my elution was not concentrated enough.  The bulk assay showed no noticeable change in the fluorescent signal of the DNA throughout the titration.  Because of the low concentration of FANCJ in the elution a second round of transfections was begun from the cell line that I had been continuing since the last transfection.  The first transfection had used the last of the lab’s stock of the pcDNA3 plasmid that contained the code for the flag tagged FANCJ.  The first step in this second round of transfections was to inoculate cultures of E. coli containing the plasmid and then purify the plasmid to make more stocks.

Working with the E. Coli was a very familiar experience since I spent most of last summer working in a lab that was attempting to create a process for manufacturing BDO in E. coli; unfortunately the first round of E. coli cultures did not grow properly and so I had to reinoculate.

The setbacks this week were frustrating but I still find research to be interesting and enjoyable.  The setbacks are just new problems to be tackled and solved.