9 thoughts on “C48 – Olson

  1. In what ways would you do this experiment differently if you had to do it over again in order to get more conclusive results?

    1. Unfortunately these results could be indicative of a range of issues including poor primer annealing, low gene expression under both conditions, and potential concentration issues with our controls. While replicating the experiment could be helpful to sort out experimental errors, other techniques could be more useful to rule out low gene expression. Part of ruling out the lack of bands due to low expression is that other groups observed expression of this gene under these conditions, but it was at a fairly low level. In the future it may be more useful to use quantitative PCR instead of end-point PCR to get a more accurate understanding of the expression of this gene. Additionally, repeating this experiment with a different primer could be useful to rule out any potential issues with the primers causing lack of bands. Finally, just careful repetitions of this experiment could be useful in trying to rule out any concentration inconsistencies.

  2. Since your primers did not anneal to the expressed gene DNA, what do you think you could do differently in the future?

    1. Ultimately, it comes down to primer selection, and selecting primers that have the highest probability of annealing to the gene of interest. This probability depends on a set of various statistics regarding the likelihood of the primer forming secondary structures, its stability, and other factors that influence how likely it is to anneal. In selecting our primer, we tried to select the primer that best fit these criteria, but our primer was still not observed to have resulted in specific annealing. Because of this, we ended up using a validated primer that other groups studying the same gene had observed specifically annealing to this gene. In the future, further experimentation with various primer sets could help us find a better suited primer to ensure that it successfully bound to our gene of interest.

    2. Ultimately, this comes down to primer design and selecting primers that have the highest probability of annealing to the gene. This probability is based on the likelihood of the primer forming secondary structures, its stability, and other factors that influence its ability to anneal. We attempted to select a primer that satisfied as many of these criteria as possible, but we still did not observe it annealing specifically to our gene. Because of this, we used validated primers, which were primers that other groups studying the same gene had found to anneal specifically, for the other experiments in the project. In the future, testing additional primer sets could be useful to find a more ideal primer.

  3. How would you adjust your lab practices to get primers that would anneal to expressed gene DNA? Is there anyway you would adjust your protocol to ensure useful results?

    1. In order to get better primer annealing, it would be beneficial to test multiple primer sets to see which one provided the most specific annealing. Getting specific annealing also depends on primer characteristics such as formation of secondary structures, stability, and other factors. In order to get a more effective primer sets, it may be useful to resasses these metrics to ensure that primers with the highest likelihood of annealing are selected.

  4. What are some of the factors that you think may be leading to your group observing a lower presence of the gene than what you had anticipated?

    1. There could be several factors leading to the lack of expression observed in our gel including primer issues, low gene expression in these conditions, or issues with the concentrations of our samples. Since we used validated primers, which were primers that other groups used and saw annealing with, it most likely that the primer was either not added or not added in sufficient quantity rather than an issue with the primer annealing specificity. Also, since other groups found our gene of interest to be expressed in these conditions, it’s unlikely that the gene was completely not expressed. However it was expressed at a low level, so it is possible that our gene is not highly expressed under these conditions for some reason. We could have also had issues with consistency of the concentrations of our samples which can lead to a lack of bands. To rule out some of these possibilities it may be necessary to repeat this experiment as well as use other methods such as quantitative PCR to gain a better understanding of our gene.

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