Could you go into a little more detail about why in the future directions section you think that fluorescence microscopy could be used to determine whether Twi8 colocallizes?
If we attached a fluorescent marker to the Twi8 protein, we could image where Twi8 goes in the cell. If we found that after inducing DNA damage, Twi8 was found more prominently in the nucleus, we would know that Twi8 colocalized with sites of DNA damage.
This experiment doesn’t really tell us what exactly Twi8 does, just that it probably has some role in repairing DNA after its been induced. We’d have to do more experiments to really know what Twi8 is doing.
There are other methods to study gene expression. RT-PCR measures mRNA expression, but another technique like western blot will measure expression by detecting the presence of the actual protein of interest. RT-PCR however is a more straightforward protocol than western blot and was probably much cheaper to do.
how many different DNA repair pathways are there? can sRNA and their associated PIWI proteins offer anything other than DNA repair? do different PIWI proteins work in conjuction to help repair DNA strand breaks?
We used a website called primer3plus, which takes a gene’s sequence and generates primers for you that are somewhere within that gene. We then took the results of that website and used BLAST, which determines if that set of primers would bind only to our gene of interest and not anywhere else – important for avoiding nonspecific or undesired amplification.
curesymposium.org 
Could you go into a little more detail about why in the future directions section you think that fluorescence microscopy could be used to determine whether Twi8 colocallizes?
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If we attached a fluorescent marker to the Twi8 protein, we could image where Twi8 goes in the cell. If we found that after inducing DNA damage, Twi8 was found more prominently in the nucleus, we would know that Twi8 colocalized with sites of DNA damage.
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Based on your conclusion that Twi8 plays an active role in repairing DNA damage, does that help you make a prediction about what the role would be?
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This experiment doesn’t really tell us what exactly Twi8 does, just that it probably has some role in repairing DNA after its been induced. We’d have to do more experiments to really know what Twi8 is doing.
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You say you chose to use that method, are there others? If there are, why was that the method you decided on?
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There are other methods to study gene expression. RT-PCR measures mRNA expression, but another technique like western blot will measure expression by detecting the presence of the actual protein of interest. RT-PCR however is a more straightforward protocol than western blot and was probably much cheaper to do.
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how many different DNA repair pathways are there? can sRNA and their associated PIWI proteins offer anything other than DNA repair? do different PIWI proteins work in conjuction to help repair DNA strand breaks?
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How did you design your primers?
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We used a website called primer3plus, which takes a gene’s sequence and generates primers for you that are somewhere within that gene. We then took the results of that website and used BLAST, which determines if that set of primers would bind only to our gene of interest and not anywhere else – important for avoiding nonspecific or undesired amplification.
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