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10 thoughts on “C42 – Carter”
Good job! Were there any challenges you faced doing these experiments?
Yes! Mainly I did not get conclusive results in the final experiment that was not shown in a figure. Basically we used FTT18 as a constitutive control (a gene that should show the same expression no matter what conditions the cell is under) and RAD51 as a positive control (a DNA repair protein that should increase expression in response to DNA Damage).. and so point being even though both of these controls did exactly what I wanted them to do, my final Hypr2c gene expression compared to these working controls had no data available for me to know if expression increased, decreased, or was not expressed at all. All of this to say, it was a bit challenging to run into this final result after a full semester worth of research, but I’m sure we could work this out if given more time/resources with a bit of tweaking to primers or PCR technique! Thanks for the question 🙂
Why do the primers have to be exactly 20 base pairs?
Hey Aidan, great question! Short answer, they don’t. Longer answer, 20bp was a great number to use that gave us enough specificity to where we didn’t get inaccurate amplification with more than one gene. By this, I mean lets say we used a 5-10bp primer.. the same 5-10bp might be available in 2 or more other genes in the genome in the exact same order and sequence as my gene Hypr2c, which could lead to multiple PCR products in my sample. 20bp allows for high specificity to one single target gene without being too long. On the other hand, primers too long could lead to self-annealing (higher probability of occurrences like hairpin loops etc.) and also it allows us to conduct PCR more rapidly, as it can take longer to wait for larger primers to anneal to our gene of interest. Overall 20bp turned out to be a great number to use in between, thanks for such an interesting question! (:
Hey Jake! What changes would you make to your gel electrophoresis techniques to increase the amount of expression seen?
Hi Kenya! The main change we need to make with gel electrophoresis is actually quantification technique. We did quantify the brightness of each band in the gel by the average brightness of all pixels available in the band in a picture we took, but there are more sensitive quantification techniques that I am not quite familiar with. Hopefully I can find more out about techniques involved with sensitive gel electrophoresis going forward! Thanks for the question, hope this helped answer a little bit!
Great presentation! What made you decide to look at Hypr2c specifically?
Hey there Dominique. Short answer, it was the gene assigned to me by Dr. Moore in a spread sheet! Long answer, these gene shows a highly similar expression profile to that of RAD51 (a known important DNA Damage repair protein) as it was expressed almost in unison with RAD51 during the conjugation phase (DNA damaging phase) of our model organism undergoing reproduction. The original name of this gene in ciliates database was actually TTHERM_00301770, however, after looking into this gene I renamed it to “Hypr2c” which stands for “Hypothetical protein expressed twice during conjugation”, which is the only thing we know about its behavior so far. Thanks for the question! (:
Could there be any other potential explanations as to why the protein wasn’t expressed?
Hi Thomas! I listed my best guesses in my video presentation, and I have not quite thought of better ideas since the recording of this presentation. To recap, if either the PCR reagents were not set up correctly or the cDNA template used in amplification was not created correctly, this might could cause the results seen but both are unlikely because my controls worked so well. My best guess is that expression was extremely low in both the DNA damaged cell and the untreated cell such that you could not see a potentially VERY dim band. I saw you asked why the protein wasn’t expressed.. this could be the case for unknown reasons, or like stated expression could be so low that it appears to have not expressed. Thanks for the question!