Student Research at Duke

image from PDB

image from PDB

A lot of the things that we went over during Responsible Conduct in Research seem like common sense at the first glance, but it is just surprising how often we neglect them in the research environment. One thing Dr. Mclay mentioned in his seminar really caught my attention is that most of the unethical behaviors in research is due to negligence and carelessness instead of intentional cheating. Having worked in a lab myself, I realize how easy it is to make mistake if not paying enough attention to my work, since most of the procedures in the labs are very detail-oriented.

I really enjoyed the movie, And The Band Played On. This movie definitely carries through its messages regarding ethical research conducts from many different perspectives, touching on issues like clinical experiments on human, privacy protection for patients, and the integrity of being a scientist. The protagonist in the movie, Don Francis, set up a good model for us to follow. His passion for research and his eagerness to search for the truth are the key characteristics of a good scientist.

cultures on shaker

three plates with colonies: blue white screening

incubator for ligation

Yesterday I did a ligation, trying to piece the BlueScript vector with the mutated DNA I made from PCR. I then did a transformation in the afternoon, expecting to see the colonies when I come in this morning. This morning when I check the plate, there are colony growth except all of the colonies are still very small, and their pigments are not fully developed either. I’ll have to incubate them for a couple more hours and I will find out if I’ve gotton the correct plasmids. The screening method I used is called Blue-white screening. If I have ligased my mutated DAN into the BlueScript vector, then my colonies should turn out white instead of blue. Here is how it works.

On the media plate we added IPTG and Xgal. Our vector contains the Lac operon that includes gene that encodes beta-galactosidase. The plasmid will express the amino terminus of the beta-galactosidase fragment while the host cell will express the C terminus fragment. Even though both of these fragments would not function on its own, they can function if both fragments are present at the same time by alpha complementation. If foreign DNA is inserted into the vector, N-terminus of the beta-galactosidase would not be produced, thus alpha complementation would not work. Therefore, if a foreign piece of DNA is inserted into the plasmid, no functional copy of the beta-galactosidase would be produced. IPTG is a substance that would induce the expression of Beta-galactosidase by mimicking lactose and binding to the repressor of the Lac operon. Thus, in the presence of IPTG, the repressors are inactivated and so transcription can occur. X gal is a galactoside that contains galactose. Beta-galactosidase would break the Xgal into galactose and a hydroxyindole. And the oxidation of the indole produces blue substance. If no beta-galactosidase is present, then Xgal would not be cleaved, thus the cell colony would be white.

I am looking forward to see if I get any white colonies.

Running the PCR

Agarose Gel

I spent the whole day yesterday running PCR and gels. As I mentioned previously, I didn’t have a successful clone so I started over. The PCR band for the overlapping fragment turned out wonderful! And my next step would need to CAREFULLY purify the DNA by cutting the bands. I’ll have to think of a way to optimize my yield for the overlapping DNA fragments.