Second Week Blog

Week 2 Daily Dues

    This week, I personally made a gel and loaded it, and learned more about enzyme restriction/ligation, overlap, plasmid extraction, and PCR. At the beginning of the week, we started growing more e. coli with tet and did some plasmid extractions to get the tet from the e. coli. We also started the Hilgarth and Anna(I don't happen to have the Anna protocol) reactions over again to figure out which reaction worked better for us. I didn't do any of the reactions myself, so I'll go over what I did learn. 


PCR

    PCR is the process of making more of a certain section of DNA. You do it through three steps, the first being denaturing, the second annealing, and the third step extension. The first step, denaturing, starts by breaking down the DNA. It does this by breaking the bond of the double helix, and sort of unwinding it. You start this step at 94-95 degrees Celsius to break it down, but change the temperature at the second step, annealing. Annealing is when your primers attach to the DNA to bind with it before the third step, extension. The difference in temperature is variable though, as different strands bind at different temperatures. This is because adenine and thymine bind at a lower temperature than cytosine and guanine, and thus, you must average the temp of them to get your annealing temp. The next step, extension, is done at 72 degrees Celsius, and is when the DNA replicates. By the end of this step, however, you are finished with your product and should have some amplified DNA. 


Theory

    Because I went over overlap last week, I'll go over more what enzyme restriction/ligation is this week. Enzyme restriction/ligation is different from overlap in the way it combines the DNA. Overlap combines DNA through having the base pairs of a left and right fragment bind with the base pairs of the middle fragment. Enzyme restriction/ligation does it by breaking apart the DNA around a sequence of palindromic nucleotide pairs. It must be a palindrome because the enzyme that binds with the DNA needs to be able to bind with the base pairs. Once the DNA is split apart by the restriction enzyme, there are primers at the ends of each side of the DNA (5' and 3') that bind with DNA liagase to sort of stick the DNA back together. 


Closing Thoughts

    By the end of the week, we concluded that the Hilgarth protocol wouldn't be followed any longer (mainly because of the touchdown PCR), and we'd be following the Anna protocol from here out. Beforehand, the samples of left, right, and tet were gathered by using a little from each protocol, but with this new set of samples, we'll only be progressing using the Anna protocol. 

Kieran

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