Mastering the Art of DNA Cooking with Alexandra Zeldenrust

    Took Friday off to do some reading and to chill out from a lot of work over the week. But going back to the lab today, I started to finish the process of 'cooking' my DNA. Again, I spent about 5 hours in the lab, and tomorrow looks like an even longer day of preparing agar gels and electrophoresing the samples of DNA to make sure I've done everything right. All this work, and I wont be able to tell until tomorrow if I did it right, or if there was any DNA on all the swabs.    Here was the recipe for today:
 All the DNA samples from Thursday are now in 500 microliter PCR tubes. I then had to label new 50 new PCR tubes and add the following to each:
      1 micro L of mitochondrial primers Forward and Reverse (these will read the strands of DNA that are going to be processed during PCR. The Forward primer will read the reference strand and the Reverse primer will read the complementary strand).
      12.5 micro L of PCR mix, which is a really cool green color.
      2 micro L   of the DNA sample
      9.5  micro L of water
      20 micro L of mineral oil to act as a protective layer that floats on top of the DNA mix to keep everything from evaporating during the 'cooking' process in the Thermal Cycler.

        Now I had to devise a clever way to mark which tubes had already had these 'ingredients' added to them because I tried just trying to sequentially putting things in, but the eventually led me to get lost in the maze of samples. This eventually led to a ritual of twisting the tubes a certain way, closing and opening caps, and by the end of this afternoon, I feel a little OCD because of it, but it worked! And I only had to redo one tube completely ( and that was due to a micropipette malfunction). After all of this was done, I centrifuged everything and placed everything in the Thermal Cycler.

       The thermal cycler is a daunting machine. It's basically a Crockpot on Steroids, PCP, and Crack. The thermal cycler 'cooks' the DNA, and using the 'ingredients' I added to each tube, it amplifies parts of the DNA that I want, mainly the mitochondrial DNA (hence the primers: Forward and Reverse). There's a block with holes for the tubes to go in it, the block heats and cools really quickly, and you have to program the steps you want it to take. For my mitochondria the steps were as follows:
            1. Two minutes at 94 degrees C to denature the DNA
            2. 30 Seconds at 94 degrees C.
            3. 40 seconds at 58 degrees C to allow the primers to find and bind to their complementary strands of DNA.
            4. 30 seconds at 72 degrees C, while the polymerase makes the complements.
            5. Repeat steps 2-4   31 times
            6. 4 minutes at 72 degrees C to allow any unfinished molecules to finish replicating.
            7. Refrigerate at 4 degrees C until needed (which will be 18 hours, as I started this process at 5:40 and would not be in the lab until 1, and I can interrupt this process).

Long day in the lab. Tomorrow will be even longer. I need to make gels to electropherese  all of these samples and if I made any mistakes I need to redo those samples. Ugh.