Monday, July 28, 2014

Katherine Vaccaro's Internship, Summer 2014 - Part 5

Hello everyone!

Last week was a busy one. After lots of troubleshooting, we finally got results for our single cell ciliate isolates! 
TA-DA! Ciliate DNA bands in gel 
6 out of 8 cells worked (not including our positive sample) which is fantastic news! I cut the bands out of the gel and saved them in individual tubes. Later, when we have a few more samples, we will be purifying the samples to extract the DNA out of the gel for cloning and transforming. And if all goes according to plan, we will be sending out our samples for sequencing and I will have concrete results telling me the species of each ciliate I picked out!

Identifying our ciliates is especially exciting, since Marie Head and I came across an interesting organism while searching our samples for ciliates:
Mr. Mohawk, Unidentified 
Meet our strange little unknown friend! I’ve been calling him “Mr. Mohawk”. Very scientific, I know. After staring at him through the microscope for several minutes, Marie and I decided he had some tintinnid-like qualities and we grabbed him for DNA amplification. Worth a shot, right? When all was said and done, we had 6 out of 8 ciliate samples work, like I said before. The cool thing is, this guy was one of them. The band he produced was bright and clear (Band on the top right of the image of our successful gel)!

When talking to Dr. Snyder about this little guy, he suggested that perhaps he is an invertebrate pluteus (larvae) that had ingested a ciliate, resulting in ciliate DNA being amplified during PCR, but he agreed that it is entirely possible to have found a previously undiscovered ciliate! Marie and I are in the process of asking around our department to see if anyone has any insight into what this little organism might be. We sure are hoping that he is a ciliate, and we are very curious to see what sequence comes back.

So have I assisted in discovering a new species of tintinnid ciliates, or have I just found a (super cute) invertebrate pluteus who had a ciliate snack before we grabbed him from our water samples? Either way, I was part of a fun little mystery, and I got to play detective while trying to determine our little mohawk friend’s identity! Fingers crossed for a new species :)

Unfortunately, my internship ends in two weeks, so I won’t be able to report back to Deep-C on the results of “The Mystery of Mr. Mohawk”, but I still attend school here at UWF so I can continue to assist Marie and Dr. Snyder even after my time with Deep-C ends.

My time here in the lab has flown by! It seems like only a few weeks ago I was getting ready for our research cruise. Now I am getting ready to say goodbye! I still have 1 more blog post to write before my time is up, so stay tuned for my last glimpse into the world of an intern in Dr. Snyder’s lab at UWF!

Posted By:
Katie Vaccaro, University of West Florida

Herbert Johnson's Internship, Summer 2014 - Part 4

Hello everyone!

I have been conducting a primary literature search gathering data on the magnitude of nitrogen fluxes to and from the Gulf of Mexico to characterize the nitrogen budget in the Gulf of Mexico. I will also use the data visualization program Ocean Data View (ODV) to plot water column profiles of nutrient distributions to gain insight into the location of the sources and sinks of nitrogen to and from the Gulf.

I hope that you all are learning a lot and having a great experience with your internships!!!!

Posted By:
Herbert Johnson, 
Florida State University

Thursday, July 24, 2014

Cynthia Kane's Internship, Summer 2014 - Part 2

I can’t believe the ten weeks are coming to an end! It seems like just yesterday I was on the boat collecting samples! The last blog post ended with PCR and gel electrophoresis, so I’ll just pick up from there. After the gel was run successfully, the bands of DNA were cut out of the gel using a scalpel and stored in the freezer. When they were ready to be used, the samples were cleaned with a DNA cleaning kit called Qiagen. Basically, the kit was used to dissolve the gel, so that only the amplified DNA was left.
DNA Cleaning Kit
After the DNA had been cleaned, it was cloned and transformed, in preparation for making clone libraries. The DNA was first mixed with a vector and a salt solution (to maintain the pH) and allowed to incubate at room temperature to allow the cells to replicate. The solution was then added to electrically competent E. coli cells and then shocked to induce the uptake of the vector by the cells. Afterwards, the solution was incubated at 37 degrees Celsius (human body temperature) for an hour, and then the solution was plated on agar plates for the bacterial colonies to grow overnight. In order to select for only the transformed colonies, ampicillin, kanamycin and x-gal were added to the gel plates. Only cells that had taken up the vector would be able to grow on the plates, as the vector contained ampicillin and kanamycin resistance, while normal E. coli cannot grow in ampicillin or kanamycin. The x-gal allowed further selection of colonies by causing colonies that did not transform properly to appear blue/purple, rather than white.

The cloning and transforming part of this process gave me a lot of trouble, as many of the samples that I plated did not form colonies during the overnight incubation. In fact, only samples 16 and 36 developed enough colonies to be transferred to well plates. The cause of this difficulty remained a mystery for several weeks. We thought originally that it was the transformation kit that I was using, so I switched to a new kit. We tried using different agar plates, in case something had gone wrong in the preparation of the plates. When this still did not solve the problem, I used chemically competent E. coli cells instead of electrically competent ones, to see if there was a problem with the machine used to shock the cells. I reused two samples that had not previously worked, samples 28 and 34. After the 18-hour incubation, it was found that all six plates (three for each sample) had grown colonies!

Top left: me plating the transformed cells
Top right: a successful cloning reaction (sample 16); the white dots indicate successfully cloned and transformed cells, the blue dots indicate cells that did not take up the vector. 
Only white colonies were used.

The final step in the DNA analysis was to prepare the DNA for sequencing. To do this, a well plate of 96 wells was used, and one colony was carefully removed from the plate with a toothpick and placed into each of the wells. LB media was added to the wells (a solution to encourage the colonies to grow more overnight). The well plate was incubated at 37 degrees Celsius for 12 hours and then divided into three shipping well trays. A 50% glycerol solution was added to these trays to act as a buffer for the DNA during transport, and they were then sealed, labeled, and sent away for sequencing.

The empty well plate, along with the agar plates containing the transformed colonies
Me pipetting the LB media into the well plates
 The well plate in the incubator. The toothpicks were removed after one hour and the well was sealed with a tape cover for overnight incubation.
Unfortunately, due to the problems we had with creating the clone libraries, the sequencing for the samples will not get to the lab until after I leave. While this is disappointing, as it would have been nice to see the sequences firsthand, I understand that this is also an important part of research; learning how to deal with challenges such as this, which are just another part of research. Also, Joe Moss, my lab instructor, said he would send me the sequences when they are finished, so that I can see the fruit of my labors!

Posted By:
Cynthia Kane, University of West Florida 

Wednesday, July 23, 2014

Erica Levine's Internship, Summer 2014 - Part 3

As this internship draws closer to its end, it’s interesting to look back at where we started and where we've come. We were able to start with a relatively unorganized collection with no form of digital record and make it into the phylogenetically organized and cataloged collection it now is. We have almost completed entering all of the jars into the digital database, and should have all 500+ previously identified items entered by the end of the week. With our remaining time, we are using genus and species keys to identify and label new specimens as they come into the collection. We will also start cataloging the specimens that are stored in larger containers and tanks too big for the shelves.

While there is still some work to be done to catch up with the identification and cataloging of all of the collected specimens, the work we have accomplished over the past month will make it much easier for others to continue expanding the collection and find items already stored at the FSU Coastal and Marine Lab. While there were a few snags along the way, working to overcome the problems and get as far along as we have has allowed me to learn new things about working in a marine collection as well as about general problem solving skills. I have enjoyed my time in this collection and look forward to any future opportunities to continue used the skills I developed while working on this internship.

Posted By:
Erica Levine, Florida State University