Week Five

I was able to use all of the training that I have been receiving over the past few weeks to officially start my own project, by testing treatment possibilities for ADLD. My work will be the first trials, so I am conducting my experiments with cell cultures. Later, if the treatments prove to be effective, there can be pre-clinical trials of the treatments run on our mice and then clinical trials with human subjects, much later on.

Many lab protocols have unavoidable waiting periods within them, and some must be performed at certain time intervals to be successful. With many molecular biology procedures, the waiting periods are either short or quite long, so they are easy to simply wait for or to start a new procedure during, so downtime can be avoided as long as there are enough tasks to be completed. Western Blotting is a prime example of this, as there are many short and long waiting periods within the overall process. The HEK 293 cells I work with needed 6 days to get through the entire treatment protocol, so I went into the lab on Saturday to complete the last part of the protocol. Though many procedures can be scheduled to fall within typical Monday-Friday work hours, not everything will and especially cell work may fall outside of regular time because cells that are currently in culture need to be dealt with around every two or three days, if not more often. For the experiments I was conducting, I needed to know the concentration of cells so I could put a certain amount of cells into each section to be treated. To find the concentration, I collected a small sample of the cells and mixed the sample with trypan blue, a stain that only can enter cells that are dead to allow differentiation from those that are alive. I then used a machine that counted the cells for me, and since trypan blue was used, the machine also compared the amount of alive cells to those that were dead.

Figure 1. Trypan blue staining, in which the blue stained cells
are dead and those that are clear are still alive. (NeoGenesis).

I was able to continue some work in bioinformatics by designing primers for future real-time PCR use. I have always found designing primers to be an extremely rewarding aspect of bioinformatics because once designed, the primers are ordered and used in the lab. The original sequence I needed was retrieved from UCSC's Genome Browser. I generated primers for that sequence using IDT's PrimerQuest Tool and then tested them using UCSC's In-Silico PCR Tool to decide exactly which to order.

Figure 2. UCSC's In-Silico PCR Tool as of July 2016. 

In-silico PCRs are online programs that can take the input of forward and reverse primer sequences as seen above (Figure 2) and generate the sequence of DNA that those primers will amplify. It was also important to find primers that would amplify a section of DNA that would include introns, since real-time PCRs use cDNA - complimentary DNA generated from RNA through reverse transcription - and we need to be able to determine if there is any gDNA - genomic DNA that transcribes into RNA - left over.

Figure 3. Example result from UCSC's In-Silico PCR.

I continued to practice certain techniques such as Western Blotting as well as helped with necessary tasks such as various PCRs/gels. One of the PCRs worked so well that the graduate student just stopped for a second in awe to look at the imaging of the gel, the lines so clean. It is simply incredible to be around people who are passionate about their work and can be so excited when things go well.

References:

NeoGenesis. n.d. LUNA. <http://www.neogenesis.co.kr/sub/sub2_view.php?bo_table=board2_1&wr_id=4>.

The bioinformatics programs and tools mentioned above can be accessed by clicking on their titles.