We believe that our primers were (for lack of a better word) good enough. Finding primers that anneal consistently can be challenging. Another option was we didn’t add our primers to the PCR tubes, but we can not be sure of this as our designed primers did not have primer dimers (meaning they attached together during PCR). Overall we think that these primers where not well suited for annealing to this gene.
What specifically does the RSP1 gene not annealing show us about the gene itself? The quantitative analysis reveals it’s likely to be involved in DNA repair but what about it not annealing does the qualitative evidence show us?
We created our own primers that should have annealed to the RSP1 gene. These designed primers not annealing means that we would get no amplification during PCR and therefore not be able to determine how the RSP1 gene was regulated during DNA damage. That is why validated primers (known to bind) were used to make sure we were able to determine how the RSP1 gene was regulated during DNA damage.
We would have used the designed primers for the RSP1 gene. instead of using the validated primers, we would have used our designed primers for the rest of the experiment. Same steps but different primers for amplification during PCR and RT PCR.
running the gel again in Figure two most likely just validates that we are seeing a larger upregulation in damaged DNA cells compared to non-damaged DNA cells. This would also potentially have an effect on the quantitative analysis as this is the image we used in the ImageJ and would therefore either be slightly different numbers potentially larger or smaller results. The reason is that the well size is about 2x larger than the band size for the untreated cells re-running it would let us have solid gels with no slight errors. we still expect the same outcome as other groups that used the same validated primers, and RSP1 gene saw an upregulation in this gene as well.
curesymposium.org 
You mention that unfortunately that RSP1 did not anneal, why do you think that it was unable to anneal, what would you do differently?
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We believe that our primers were (for lack of a better word) good enough. Finding primers that anneal consistently can be challenging. Another option was we didn’t add our primers to the PCR tubes, but we can not be sure of this as our designed primers did not have primer dimers (meaning they attached together during PCR). Overall we think that these primers where not well suited for annealing to this gene.
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What specifically does the RSP1 gene not annealing show us about the gene itself? The quantitative analysis reveals it’s likely to be involved in DNA repair but what about it not annealing does the qualitative evidence show us?
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We created our own primers that should have annealed to the RSP1 gene. These designed primers not annealing means that we would get no amplification during PCR and therefore not be able to determine how the RSP1 gene was regulated during DNA damage. That is why validated primers (known to bind) were used to make sure we were able to determine how the RSP1 gene was regulated during DNA damage.
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If the RSP1 did anneal, what would your next steps have been?
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If the RSP1 did anneal, what would your next steps have been?
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We would have used the designed primers for the RSP1 gene. instead of using the validated primers, we would have used our designed primers for the rest of the experiment. Same steps but different primers for amplification during PCR and RT PCR.
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Do you think that running the result in Figure 2 with a new gel/wells would yield a different result in terms of the bands expected length?
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running the gel again in Figure two most likely just validates that we are seeing a larger upregulation in damaged DNA cells compared to non-damaged DNA cells. This would also potentially have an effect on the quantitative analysis as this is the image we used in the ImageJ and would therefore either be slightly different numbers potentially larger or smaller results. The reason is that the well size is about 2x larger than the band size for the untreated cells re-running it would let us have solid gels with no slight errors. we still expect the same outcome as other groups that used the same validated primers, and RSP1 gene saw an upregulation in this gene as well.
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For you, what was the most difficult part of this project and why?
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