For the major Health & Life Science we have a practical every block. During the practical we get lab experience. We practice our pipette skills, wear lab coats, and do awesome things! This time we spent multiple days in the lab because the practical was long and there were a lot of steps that we needed to complete. But the practical can be divided into 3 large steps.
Step 1: Ligation
We want to look at a specific protein and where it is expressed. To do this we need to add a fluorescent marker to our protein. So what we did was sort of glue pieces of DNA together. We have a plasmid (a little piece of circular DNA, like bacteria) with the fluorescent marker, we cut it open and add the piece of DNA for the gene we want to look at. We then have a circular DNA with the gene for the specific protein, the fluorescent marker which is added to the protein, and all the other things to make sure the protein is produced in normal cells.
Step 2: Transformation
Then, to get large amounts of the DNA we integrated our plasmids into E. coli bacteria. These bacteria then replicated our DNA for us. E. coli is a great bacteria to use because it divides every 20 minutes, doubling the DNA. This means that after a day your initial amount of DNA has grown exponentially. The bacteria were grown in petri dishes over night and the next day it was time for use to extract our plasmids. This takes multiple small steps but the idea is that you dissolve the bacteria and isolate the DNA plasmids with the fluorescent marker and the specific protein. Then these DNA plasmids continue to the next step.
Step 3: Transfection
Here the DNA plasmids are introduced to Human Embryonic Kidney (HEK) cells using multiple small steps. These HEK cells then integrate the plasmids into their DNA and start producing a fluorescent protein (the one we want to see under the microscope). After allowing the cells to grow and work for a day we checked them. We used a fluorescence microscope and could see the protein, which was fluorescent, in the cells.
Type to you later,