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 

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