Question Asked: you mentioned that there was no band and you have to redo the experiment. Do you think there are any experimental errors that could have caused this, if so what?
A very small amount of cDNA is added to the PCRs, and if it is left around the top of the microcentrifuge tube instead of being at the bottom it could evaporate. As this one of of the last PCRs we did, we believe that the cDNA could have evaporated which is why we did not see a band.
We believe that when our PCRs were set up, cDNA was not pipetted directly to the bottom of the microcentrifuge tube. As it was one of the last PCRs done, the small amount of cDNA left around the top of the tube could have evaporated. This means that we don’t know if our -RT were correct or if the same thing occurred. This also means we could not fully concluded if lack of expression of TWI7 was because it is not involved the DNA damage DS repair pathway or it was because DNA damage was not induced.
gDNA is the DNA of the genome and is transcribed at all times. If primers anneal to that DNA (as ours did) there should always be a band. cDNA, however, is the DNA of the transcriptase. Therefore, we expected to see an increase in the cDNA if that gene was being made more when DNA damage has been induced.
Hydroxyurea causes DNA polymerase to slow down which decrease dNTPs being added to DNA. This slowing of the replication fork causes a lot of instability making the fork collapse. This results is Double Stranded DNA breaks. As hydroxyurea is very strong there are many breaks.
Hello, amazing work! Is there anything you would change or specifically test for when you redo RT-PCR to determine if cell damage was induced or not, or if TWI7 is involved in the pathway or not? Thanks!
Hydroxyurea is a very strong DNA damaging agent so I would keep that the same. However I would be more careful to pipette all regents for one PCR at a time to ensure nothing gets degraded. Further study could look at different kinds of DNA damage repair pathways such as Single-Stranded Repair Pathway.
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Question Asked: you mentioned that there was no band and you have to redo the experiment. Do you think there are any experimental errors that could have caused this, if so what?
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A very small amount of cDNA is added to the PCRs, and if it is left around the top of the microcentrifuge tube instead of being at the bottom it could evaporate. As this one of of the last PCRs we did, we believe that the cDNA could have evaporated which is why we did not see a band.
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Question: Why do you think no band was present in the RAD51 positive control, and how did that effect the results that were on the rest of that gel.
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We believe that when our PCRs were set up, cDNA was not pipetted directly to the bottom of the microcentrifuge tube. As it was one of the last PCRs done, the small amount of cDNA left around the top of the tube could have evaporated. This means that we don’t know if our -RT were correct or if the same thing occurred. This also means we could not fully concluded if lack of expression of TWI7 was because it is not involved the DNA damage DS repair pathway or it was because DNA damage was not induced.
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Question: what is the difference between cDNA and gDNA, and why was there a differnce between the 2 on your gel in figure 1?
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gDNA is the DNA of the genome and is transcribed at all times. If primers anneal to that DNA (as ours did) there should always be a band. cDNA, however, is the DNA of the transcriptase. Therefore, we expected to see an increase in the cDNA if that gene was being made more when DNA damage has been induced.
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What is the general mechanism in which hydroxyurea intervention causes DNA damage?
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Hydroxyurea causes DNA polymerase to slow down which decrease dNTPs being added to DNA. This slowing of the replication fork causes a lot of instability making the fork collapse. This results is Double Stranded DNA breaks. As hydroxyurea is very strong there are many breaks.
LikeLike
Hello, amazing work! Is there anything you would change or specifically test for when you redo RT-PCR to determine if cell damage was induced or not, or if TWI7 is involved in the pathway or not? Thanks!
LikeLike
Hydroxyurea is a very strong DNA damaging agent so I would keep that the same. However I would be more careful to pipette all regents for one PCR at a time to ensure nothing gets degraded. Further study could look at different kinds of DNA damage repair pathways such as Single-Stranded Repair Pathway.
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