|DNA Cleaning Kit|
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.|