Thank you for the question Rebecca! The reason why we used different concentrations of agarose between the two gels is because we had different expected band sizes in the two gels that we ran. The higher the agarose %, the slower the DNA will migrate down the gel. We used a smaller (1.2%) concentration on one of the gels because we had larger DNA expected bp lengths and in doing this, we had more efficient movement of the larger DNA products. It is kind of confusing, so I hope this answer made sense.
There could have been multiple reasons as to why no band showed up in this lane. We could have forgotten to add cDNA into the PCR tube for this Loading Control lane. We also could have forgotten one, or both of the Rpp0 primers. It could have also been due to inadequate pipetting into the agarose gel for this lane.
Great question! I think it would be important to redo these studies on a bigger scale without the limitations of the specific RT-PCR that we ran. Once those are done quantitative analyses can be carried out upon those gels to see how big of an increase there is in Rdn2 before and after damage. Finally cellular fractionation can be done on Rdn2 to find the localization of the gene within the cell!
Rdn2 itself is a protein that becomes more robust, or more highly recruited, when DNA damage occurs within a cell. Nothing in these studies necessarily make cells more robust, but instead DNA damage makes the Rdn2 protein more robust because it is a necessary protein in the DNA damage repair pathway. Hope this answer makes sense!
curesymposium.org 
why did you run the primer validation gel on a lower 1.2% agarose gel than the 1.5% DNA damage gel? is there a reason for that difference?
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Thank you for the question Rebecca! The reason why we used different concentrations of agarose between the two gels is because we had different expected band sizes in the two gels that we ran. The higher the agarose %, the slower the DNA will migrate down the gel. We used a smaller (1.2%) concentration on one of the gels because we had larger DNA expected bp lengths and in doing this, we had more efficient movement of the larger DNA products. It is kind of confusing, so I hope this answer made sense.
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Why do you think the band didn’t show up in the control lane?
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There could have been multiple reasons as to why no band showed up in this lane. We could have forgotten to add cDNA into the PCR tube for this Loading Control lane. We also could have forgotten one, or both of the Rpp0 primers. It could have also been due to inadequate pipetting into the agarose gel for this lane.
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If your results are confirmed what other research might you want to do on Rdn2?
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Great question! I think it would be important to redo these studies on a bigger scale without the limitations of the specific RT-PCR that we ran. Once those are done quantitative analyses can be carried out upon those gels to see how big of an increase there is in Rdn2 before and after damage. Finally cellular fractionation can be done on Rdn2 to find the localization of the gene within the cell!
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Does this mean that Rdn2 makes cells more robust after repair than they were before degradation and repair?
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Rdn2 itself is a protein that becomes more robust, or more highly recruited, when DNA damage occurs within a cell. Nothing in these studies necessarily make cells more robust, but instead DNA damage makes the Rdn2 protein more robust because it is a necessary protein in the DNA damage repair pathway. Hope this answer makes sense!
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