We believe there was no gene expression in our DNA damage response for our gene because it wasn’t involved directly in DNA damage response regardless of the similar pattern of expression against known genes of DNA damage response, we were skeptical at first but this was corroborated with other groups studying our same gene. Without that additional confirmation, we wouldve liked to redo the gene expression PCR and gel imaging just to fully ensure we didnt generate any user errors when setting up and running the experiments!
Thanks for the question!
We believe there was no gene expression for our experimental gene because our gene is not directly included in DNA damage repair, so there would be no change in expression upon inducing damage. We were skeptical at first considering the similarity in the gene expression pattern between our gene and known DNA damage repair genes, but other groups studying the same gene reached the same conclusion so it put us a little more at ease with the result. we would’ve liked to redo the PCR’s and gel for gene expression to ensure that the results didn’t vary at all and possibly just clean up our data, but we didn’t have the time to do so!
Thanks for the Question!
Since we used two different set ups for PCR’s, the first being a primer validation PCR to ensure that our primer set we believed would anneal to our genome based on the sequence we were able to derive, the controls for that PCR were: Negative control was mixing H2O with both the primers which we know would anneal to ATPVOC6 and the primer set we designed in separate lanes, and since theres no DNA present in H2O we didnt expect to see any results in those two lanes on the rightmost edge of figure 2 in results(except for possible primer dimers showing at the very bottom of the gel. and for that our positive control was ATPVOC6 primers mixed in with out cDNA and gDNA samples in sample lanes 3/4 of figure 2, we know that ATPVOC6 is always expressed in the T. thermophila so we expected to see bands as we got in results.
As far as our gene expression images (3a/3b) our negative controls were samples that didnt contain any reverse transcriptase(the samples with the -RT indication in the label), as without any reverse transcriptase, there should be no visible bands for transcription. our positive controls for this gel were both the lanes for Rad51 expression and ATPVOC6 expression, as we know rad51 is involved in DNA damage repair, we should expect to see a band of increased brightness to match the increased expression to repair the sequence which we saw as a result, and the ATP lanes since the ATP gene is always expressed, we expected to see no difference in expression levels across untreated and DNA damage samples and we got that result too.
Thanks for the question!
I dont know if the images are popping up in the link for my poster but from the presentation:
Figure 2:(negatives in lanes 5/6) (positives in lanes 3/4)
Figure 3a: (Negatives in lanes 3/4 +7/8 after the ladder) (Positives in lanes 1/2 + 5/6 after the ladder)
Figure 3b: (Negatives in lanes 3/4 after ladder) and the other samples were our experimental samples
Why do you think there was no expression of the gene in DNA damage response?
We believe there was no gene expression in our DNA damage response for our gene because it wasn’t involved directly in DNA damage response regardless of the similar pattern of expression against known genes of DNA damage response, we were skeptical at first but this was corroborated with other groups studying our same gene. Without that additional confirmation, we wouldve liked to redo the gene expression PCR and gel imaging just to fully ensure we didnt generate any user errors when setting up and running the experiments!
Thanks for the question!
We believe there was no gene expression for our experimental gene because our gene is not directly included in DNA damage repair, so there would be no change in expression upon inducing damage. We were skeptical at first considering the similarity in the gene expression pattern between our gene and known DNA damage repair genes, but other groups studying the same gene reached the same conclusion so it put us a little more at ease with the result. we would’ve liked to redo the PCR’s and gel for gene expression to ensure that the results didn’t vary at all and possibly just clean up our data, but we didn’t have the time to do so!
Thanks for the Question!
Great presentation! What controls were used for the PCR?
Since we used two different set ups for PCR’s, the first being a primer validation PCR to ensure that our primer set we believed would anneal to our genome based on the sequence we were able to derive, the controls for that PCR were: Negative control was mixing H2O with both the primers which we know would anneal to ATPVOC6 and the primer set we designed in separate lanes, and since theres no DNA present in H2O we didnt expect to see any results in those two lanes on the rightmost edge of figure 2 in results(except for possible primer dimers showing at the very bottom of the gel. and for that our positive control was ATPVOC6 primers mixed in with out cDNA and gDNA samples in sample lanes 3/4 of figure 2, we know that ATPVOC6 is always expressed in the T. thermophila so we expected to see bands as we got in results.
As far as our gene expression images (3a/3b) our negative controls were samples that didnt contain any reverse transcriptase(the samples with the -RT indication in the label), as without any reverse transcriptase, there should be no visible bands for transcription. our positive controls for this gel were both the lanes for Rad51 expression and ATPVOC6 expression, as we know rad51 is involved in DNA damage repair, we should expect to see a band of increased brightness to match the increased expression to repair the sequence which we saw as a result, and the ATP lanes since the ATP gene is always expressed, we expected to see no difference in expression levels across untreated and DNA damage samples and we got that result too.
Thanks for the question!
I dont know if the images are popping up in the link for my poster but from the presentation:
Figure 2:(negatives in lanes 5/6) (positives in lanes 3/4)
Figure 3a: (Negatives in lanes 3/4 +7/8 after the ladder) (Positives in lanes 1/2 + 5/6 after the ladder)
Figure 3b: (Negatives in lanes 3/4 after ladder) and the other samples were our experimental samples
How do you induce damage in the cells?