I’m not too sure if there would be any major phenotypic effects of a Twi8 knockout, but if it was knocked out, I’d expect that the cell’s response to DNA damage might be compromised. Although, if the cell’s response to DNA damage is compromised, this could result in the accumulation of necrosis, or unplanned cell death, which isn’t pretty.
Why did you choose to run a 2% agarose gel? And did you get your clear gel results you’ve displayed on the first go around, or did you have to run multiple?
We chose to run on a 2% agarose gel due to the expected sizes of our DNA products. Different electrophoresis gel concentrations can work better or worse with different lengths of DNA products.
We got our gel results on the first go around. We used a program called Fiji which allows us to edit the image so our bands show up clearly by adjusting image components such as brightness and contrast.
Hydroxyurea isn’t what is directly showing bright expression, it’s the amount of the Twi8 gene transcribed that creates the band. But, if Twi8 wasn’t showing a brighter band in the +HU column (hydroxyurea present) for our gene-specific primers (NEC section of the results figure), this would indicate that Twi8 is not involved in the DNA damage repair pathway. This result would probably warrant a gene knockout experiment to find what other role Twi8 plays instead of DNA damage.
Yes, the main research question of our experiment was to find out if our assigned gene does or does not play a role in the DNA damage repair pathway. This allowed us to form a hypothesis in which we expected the expression of Twi8 to increase under DNA damage. Our results confirmed this hypothesis. If we found that our gene did not increase in expression under DNA damage, this would mean that our hypothesis was incorrect.
First, our Rpp0 controls, or loading controls, aimed to make sure that we loaded the gel correctly.
Second, our Rad51 controls aimed to tell us if DNA damage was occurring as expected/if our hydroxyurea was functioning correctly. Rad51 is known to increase in expression under DNA damage, so if we saw a brighter band in the +HU column (hydroxyurea present), this would mean that our hydroxyurea is functioning correctly.
Our third type of control was the -RT columns present in all of the different primers used (Rpp0, Rad51, and NEC primers). The purpose of having these columns is to make sure there is no contamination in any of our samples. The -RT stands for “without reverse transcriptase” which means there is no transcription agent to create bands, which makes this a great indicator of contamination as we expect to not see any bands in those columns.
I should also mention how we found them. The Rpp0, Rad51, and -RT control methods were used by every group. We were instructed to use them by the class, but I’m not sure exactly how the methods were all discovered.
curesymposium.org 
Do you have any guesses about the potential phenotypic effects of knocking out TW18?
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I’m not too sure if there would be any major phenotypic effects of a Twi8 knockout, but if it was knocked out, I’d expect that the cell’s response to DNA damage might be compromised. Although, if the cell’s response to DNA damage is compromised, this could result in the accumulation of necrosis, or unplanned cell death, which isn’t pretty.
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Why did you choose to run a 2% agarose gel? And did you get your clear gel results you’ve displayed on the first go around, or did you have to run multiple?
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We chose to run on a 2% agarose gel due to the expected sizes of our DNA products. Different electrophoresis gel concentrations can work better or worse with different lengths of DNA products.
We got our gel results on the first go around. We used a program called Fiji which allows us to edit the image so our bands show up clearly by adjusting image components such as brightness and contrast.
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What would happen if hydroxyurea wasn’t showing such a bright expression? What would you do then?
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Hydroxyurea isn’t what is directly showing bright expression, it’s the amount of the Twi8 gene transcribed that creates the band. But, if Twi8 wasn’t showing a brighter band in the +HU column (hydroxyurea present) for our gene-specific primers (NEC section of the results figure), this would indicate that Twi8 is not involved in the DNA damage repair pathway. This result would probably warrant a gene knockout experiment to find what other role Twi8 plays instead of DNA damage.
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Was there a particular reason you hypothesized that DNA damage would increase the transcription of the TWI8 gene?
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Yes, the main research question of our experiment was to find out if our assigned gene does or does not play a role in the DNA damage repair pathway. This allowed us to form a hypothesis in which we expected the expression of Twi8 to increase under DNA damage. Our results confirmed this hypothesis. If we found that our gene did not increase in expression under DNA damage, this would mean that our hypothesis was incorrect.
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What is the Gene TWI8 control? How was it found?
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We had numerous controls in this experiment.
First, our Rpp0 controls, or loading controls, aimed to make sure that we loaded the gel correctly.
Second, our Rad51 controls aimed to tell us if DNA damage was occurring as expected/if our hydroxyurea was functioning correctly. Rad51 is known to increase in expression under DNA damage, so if we saw a brighter band in the +HU column (hydroxyurea present), this would mean that our hydroxyurea is functioning correctly.
Our third type of control was the -RT columns present in all of the different primers used (Rpp0, Rad51, and NEC primers). The purpose of having these columns is to make sure there is no contamination in any of our samples. The -RT stands for “without reverse transcriptase” which means there is no transcription agent to create bands, which makes this a great indicator of contamination as we expect to not see any bands in those columns.
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I should also mention how we found them. The Rpp0, Rad51, and -RT control methods were used by every group. We were instructed to use them by the class, but I’m not sure exactly how the methods were all discovered.
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