11 thoughts on “C36 – Thillot

  1. If the Hyplrr gene doesn’t participate in the DNA damage repair pathway, does it have other functions during DNA replication/transcription?

    1. We suspect it has a role to play in centrosome separation during early mitosis as the only close homologs to our protein were involved in this pathway.

  2. Could you explain how you went from mRNA to cDNA? How do you go through the process of reverse transcriptase? I haven’t taken this lab yet so I’m not sure how that works.

    1. Yes so we use a primer that anneals to the poly-A tail found in mRNA known as a poly-T primer and from there using our DNA polymerase it extends from the primer sequence to make a complementary strand. In this reaction is the usual components of PCR which will make multiple copies of cDNA during each round of its cycle. There will be a denaturation of the two strands, annealing of primers, and then elongation. The components of general deoxynucleotides and Mg2+ buffers are needed as any normal DNA -> RNA or DNA ->DNA conversion.

  3. Could you explain how you went from mRNA to cDNA? How did you use reverse transcriptase to get to cDNA? I haven’t taken this lab yet so I’m not quite sure how that works.

  4. You mentioned testing your primers on a gel with RPPO, what would happened if you used a different gene? How would the gel results differ?

    1. RPPO was used as a positive control because it is always expressed in T.thermophila and expressed the same under DNA damage as it is not involved in that pathway. With that said, if we used a different gene then it would need to meet this same criteria or it wouldn’t be trusted as a control making our results difficult to interpret.

  5. You mentioned in your future directions that they should use 3 different primers, do you think you didn’t get accurate results because of the amount of primers you used or the specific types of primers you used?

    1. Our anneal test showed that our primers annealed to our gene of interest well so I would think that wouldn’t be an issue, but likely degradation of our primers could be a factor. There is the fact that RNA degrades very easily, especially only a few bases as seen with primers. So overall I think the amount was an issue.

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