6 thoughts on “C33 – Smith

  1. Amazing poster and presentation! The only thing I wanted to ask was why you choose to use Rpp0 as a control? What is this? and Why is it relevant?

    1. Thank you! Yeah, so we used Rpp0 as our positive control in the RT-PCR experiment. Rpp0 is a primer that is known to anneal to the T. thermophila gene in a consistent way; this means that there will be similar expression in both the +HU (DNA damaged cells) and UT (untreated cells) cells. We needed a way to show that similar amounts of cDNA were added to both the +HU and the UT and that differences in gene expression didn’t come from just different amounts of cDNA being amplified. So we use it as a loading control and double check that band intensities are similar from the expression of Rpp0. There are other loading controls that can be used, but this was the one in our class that we had.

    1. So cDNA stands for complementary DNA and gDNA stands for genomic DNA. When we think of DNA we often think of gDNA, that is the genomic sequence found on our DNA. This is the A’s T’s G’s and C’s exactly how it is on our DNA. The thing about gDNA is it contains all the introns and exons and other noncoding regions. As we know intros are spliced out and this leads to the cDNA. The cDNA is then the nucleotides that will be transcribed into RNA and contain only the exons. In our case with our project, the Hycop gene did not contain any introns and therefore the cDNA and gDNA were the exact same length.

    1. So first we were given a gene to study, in our case this is the Hycop gene. We used online programs called Primer3Plus and BLAST. We used certain parameters to select the best primer deign. For example, we wanted the presence of G or C bases at the 3′ end. This is called a GC clamp and due to the stronger bonding of G’s to C’s (the 3 hydrogen bonds). Another parameter is cross homology, we used BLAST to make sure that the primers would then bind specifically to our sequence and not other regions of the T. thermophilia gene. All of these factored into our choice for of specific primers. The primers were then created by a company and send to us here at CU.

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