We used a program where we uploaded the sequence of the genes we were studying, called Primer3Plus, which then gave us a bunch of different primer options to amplify our gene. We then picked our primer based of certain parameters. At that point we didn’t know for sure if the primer would anneal to our gene, so we had to run a primer validation gel to make sure they could work before running our gene expression gel.
Vija, awesome presentation! I’m wondering why more sensitive assays (Northern or Western blotting) would be helpful for you in ruling out the involvement of Nuftr 1 in the DNA damage response pathway? What is different about those assays in this context? Thank you so much!
So in the gels we ran, we just tested if the gene would even be copied or amplified when there was damaged DNA present. However, Northern blotting would test if the mRNA of my gene was made, and Western blotting would test if a protein was made out of my gene in response to DNA damage. These would be more accurate for testing gene expression in this case. Since past research says this gene is involved in transcription and chromatin structure, which play some role in DNA damage repair, but it could just be that it isn’t expressed as much, and therefore we can’t see it on these gels.
We used a program called Primer3Plus. This allowed us to upload our gene sequences, which then gave us a bunch of different primer options. We then followed a bunch of parameters to find the primer that would perform and anneal the best to our gene.
We used a program called Primer3Plus. This allowed us to upload our gene sequences, which then gave us a bunch of different primer options. We then followed a bunch of parameters to find the primer that would perform and anneal the best to our gene.
Why do you think that the Nuftr 1 gene experienced a decrease in expression when exposed to DNA damage? In other words if the gene simply is not involved in DNA damage repair then wouldn’t its expression stay the same?
I think that was in part on how I quantified my data, but if not, then it probably means that Nuftr1 is likely not involved in the DNA damage response pathway.
What is the method and point of validating primers?
We used a program where we uploaded the sequence of the genes we were studying, called Primer3Plus, which then gave us a bunch of different primer options to amplify our gene. We then picked our primer based of certain parameters. At that point we didn’t know for sure if the primer would anneal to our gene, so we had to run a primer validation gel to make sure they could work before running our gene expression gel.
Vija, awesome presentation! I’m wondering why more sensitive assays (Northern or Western blotting) would be helpful for you in ruling out the involvement of Nuftr 1 in the DNA damage response pathway? What is different about those assays in this context? Thank you so much!
So in the gels we ran, we just tested if the gene would even be copied or amplified when there was damaged DNA present. However, Northern blotting would test if the mRNA of my gene was made, and Western blotting would test if a protein was made out of my gene in response to DNA damage. These would be more accurate for testing gene expression in this case. Since past research says this gene is involved in transcription and chromatin structure, which play some role in DNA damage repair, but it could just be that it isn’t expressed as much, and therefore we can’t see it on these gels.
How did you decide which primers to use?
We used a program called Primer3Plus. This allowed us to upload our gene sequences, which then gave us a bunch of different primer options. We then followed a bunch of parameters to find the primer that would perform and anneal the best to our gene.
How did you design your primers?
We used a program called Primer3Plus. This allowed us to upload our gene sequences, which then gave us a bunch of different primer options. We then followed a bunch of parameters to find the primer that would perform and anneal the best to our gene.
Why do you think that the Nuftr 1 gene experienced a decrease in expression when exposed to DNA damage? In other words if the gene simply is not involved in DNA damage repair then wouldn’t its expression stay the same?
I think that was in part on how I quantified my data, but if not, then it probably means that Nuftr1 is likely not involved in the DNA damage response pathway.