In your future directions you mention using a fluorescent tag on Soccax and known DNA damage repair proteins. Who you use the same fluorescent tags or one for Soccax and another from the DNA damage repair proteins?
Honestly, I’m not incredibly familiar with fluorescent tagging, but depending on which DNA damage repair protein studied, there may be different tags required. It all depends on which known repair protein you choose, and whether that protein and the Socax protein have functional groups/regions where the same tag can attach to. If not, different tags would have to be used.
In the first PCR test (primer validation gel), I was expecting a band of similar intensity/thickness with the cDNA as the gDNA when amplified with the Socax primers. This is because I theoretically would have loaded in the same amount of DNA template in both PCR samples, so the thickness would be the same. Also, since both gDNA and cDNA consisted of just part of one exon, I didn’t see why expression would be different between them since they both have the exact same DNA bases (because there was no intron to be spliced out for cDNA).
Sorry, I meant: since the Socax primers would only amplify part of one exon in both gDNA and cDNA, I didn’t see why expression would be different between them.
Hi, I thought this was a super interesting presentation. I was wondering if you could create a different primer that would anneal to the cDNA and how you would expect that to change your results?
Yes, we could create a different primer. Different primers were used in other groups studying the same gene, and theirs seemed to successfully anneal to the cDNA. In their primer validation gel, they saw bands of similar intensity and thickness with their Socax primer lanes with both cDNA and gDNA. I only saw a band in the gDNA lane.
Then, with the experimental RT-PCR, many of those with primers that annealed to the cDNA saw a dimmer band in the lane without DNA damage and a brighter band in the lane with DNA damage, indicating an uptick in expression. However, because mine most likely didn’t anneal to the cDNA, I didn’t see anything in those lanes.
Sorry for the confusion! My gel did not indicated up-regulated expression of Socax, but the consensus gel did. The consensus gel is what most other people studying Socax doing this same experiment found, and they found that Socax was potentially up-regulated after DNA damage.
In your future directions you mention using a fluorescent tag on Soccax and known DNA damage repair proteins. Who you use the same fluorescent tags or one for Soccax and another from the DNA damage repair proteins?
Honestly, I’m not incredibly familiar with fluorescent tagging, but depending on which DNA damage repair protein studied, there may be different tags required. It all depends on which known repair protein you choose, and whether that protein and the Socax protein have functional groups/regions where the same tag can attach to. If not, different tags would have to be used.
Hi Pauline,
Great Presentation. Can you explain again wether in the first PCR test you were expecting a brighter band in the cDNA and why?
In the first PCR test (primer validation gel), I was expecting a band of similar intensity/thickness with the cDNA as the gDNA when amplified with the Socax primers. This is because I theoretically would have loaded in the same amount of DNA template in both PCR samples, so the thickness would be the same. Also, since both gDNA and cDNA consisted of just part of one exon, I didn’t see why expression would be different between them since they both have the exact same DNA bases (because there was no intron to be spliced out for cDNA).
Sorry, I meant: since the Socax primers would only amplify part of one exon in both gDNA and cDNA, I didn’t see why expression would be different between them.
Which florescent tags would you use when comparing the presence of your gene and a known DNA damage repair gene?
Hi, I thought this was a super interesting presentation. I was wondering if you could create a different primer that would anneal to the cDNA and how you would expect that to change your results?
Yes, we could create a different primer. Different primers were used in other groups studying the same gene, and theirs seemed to successfully anneal to the cDNA. In their primer validation gel, they saw bands of similar intensity and thickness with their Socax primer lanes with both cDNA and gDNA. I only saw a band in the gDNA lane.
Then, with the experimental RT-PCR, many of those with primers that annealed to the cDNA saw a dimmer band in the lane without DNA damage and a brighter band in the lane with DNA damage, indicating an uptick in expression. However, because mine most likely didn’t anneal to the cDNA, I didn’t see anything in those lanes.
How is gel electrophoresis performed and why would we still expect brighter or more intense bands even with DNA breaks? What is cDNA?
What type of experiments would you do further to understand the gene a bit better and possibly fully prove or disprove your hypothesis.
How did your gel indicate up-regulated expression of Socax? You mentioned this around 4:05 in your presentation.
Sorry for the confusion! My gel did not indicated up-regulated expression of Socax, but the consensus gel did. The consensus gel is what most other people studying Socax doing this same experiment found, and they found that Socax was potentially up-regulated after DNA damage.