Week 7:
University of Iowa Department of Biochemistry

July 10, 2013

With the Fourth of July on Thursday of this week, we only had 3 work days followed by a 4 day weekend.  This was great because the Iowa City Jazz Festival was happening downtown where I got to see numerous musicians play some great jazz.  During the work week, however, I did prepare H28, H31 and H32 for confocal microscopy in Week 8 as well as preparing H28 and H33 for transgenic injection on Tuesday of week 8.  H28 needs to be re-injected because all of the tadpoles from the first injection lost their expression before we sectioned them.

I also started working on cloning the N-Terminus construct by creating fragments using primers Yuan ordered for me as well as the templates used to create each fragment with the vector being used for one fragment and the cDNA Yuan cloned by Reverse Transcriptase PCR using the mRNA isolated from Xenopus tropicalis brain and retina tissue.  After failing to get the desired fragments the first time around, we used touchdown PCR to increase the specificity of the fragments created.  Touchdown PCR is essentially Phusion PCR with a special beginning sequence.  The machine first denatured the DNA as usual, but then the first annealing temperature was 5 degrees C above the normal annealing temperature.  This ensures the primers will specifically anneal to where we want them to anneal and prevents random annealing to other parts of the cDNA.  However, this temperature may be too high for the primers to anneal at all, so for the next cycle, the annealing temperature is lowered by 1 degree C.  We repeat this for a total of 10 cycles with each cycle 1 degree C lower on the annealing step than the previous cycle.  Following this process, we then run the remaining part of the reactions as we would run a Phusion PCR.  Using this technique, I saw results on the gel that look like what I was hoping for and Yuan got very excited that we wouldn’t have to spend a week trying to get that to work.

The more I learn about how research is conducted at every level here at the University of Iowa, the more it becomes apparent that one of the most attractive qualities of studying at this institution is the collaborative spirit that permeates the majority of the programs.  The medical school trains PA’s alongside MD’s to prepare the PA’s to work with MD’s and vice versa.  There’s also the MD/PhD program which has its students working the MD program as well as in laboratories with the grad students.  The grad students work in their labs, but if they find something interesting and want to use the equipment or consult individuals of another lab or department, they’re encouraged to do so.  This connection between all of the research faculty and education granted to the students of this environment is really pleasing to my ideals.  People working hard and helping each other to progress scientific knowledge should be as easy as possible, and I think the U of I has a system in place that really lends itself to doing just that.