Question: What type of information do you enter into the databases you mentioned during the methods, and how does this help you determine which primers will anneal to the genes that you are interested in?
You can find the whole gene sequence in the Tetrahymena genome database, and copy and paste that into the Primer3Plus software. It will autogenerate primer suggestions based on parameters you put into the software. The most important aspects are annealing temperature, GC%, RNA instability, and the risks that it could form secondary structures. We chose the ones that best fit these parameters.
Question: Do you expect any significantly different results if you were to proceed with testing Twi7 against more natural occurrences of double stranded DNA breaks or single stranded breaks like you previously mentioned?
Not necessarily, in theory the DSBs should be similar on a molecular level; this would be more as a means to verify our results should the function of Twi7 continue to elude us. We would likely proceed by testing the function of the protein in protecting DNA from damage, or in repairing single strand breaks first.
Our lab as a whole was working with testing the expression of several genes against DSB repair pathways. This was done following prior research by Dr. Suzanne Lee at WWU indicating that when these genes were knocked out, there was an accumulation of DSBs
We would likely verify that Twi7 indeed was upregulated following the induction of Double Stranded Breaks. However, since this result does fall in line with earlier observations by Dr. Suzanne Lee at WWU that Twi7 seems to play less of a role in DSB repair, we likely will not pursue this verification.
Based on the information gathered within the databases, how were three primers materialized? Did the software create them or did it just guide you to the best primer available for use?
Since the whole Twi7 gene is available on the Tetrahymena genome database, we were able to copy and paste that into the Primer3Plus software. It can autogenerate primer suggestions based on parameters you put into the software. The most important aspects are annealing temperature, GC%, RNA instability, and the risks that it could form secondary structures. We chose the ones that best fit these parameters. The RPPO and Rad51 primers, as well as the validated primers for Twi7 are commercially available.
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Question: What type of information do you enter into the databases you mentioned during the methods, and how does this help you determine which primers will anneal to the genes that you are interested in?
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You can find the whole gene sequence in the Tetrahymena genome database, and copy and paste that into the Primer3Plus software. It will autogenerate primer suggestions based on parameters you put into the software. The most important aspects are annealing temperature, GC%, RNA instability, and the risks that it could form secondary structures. We chose the ones that best fit these parameters.
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Question: Do you expect any significantly different results if you were to proceed with testing Twi7 against more natural occurrences of double stranded DNA breaks or single stranded breaks like you previously mentioned?
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Not necessarily, in theory the DSBs should be similar on a molecular level; this would be more as a means to verify our results should the function of Twi7 continue to elude us. We would likely proceed by testing the function of the protein in protecting DNA from damage, or in repairing single strand breaks first.
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Why did you analyze the break sites of double-stranded DNA instead of the break sites of single-stranded DNA?
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Our lab as a whole was working with testing the expression of several genes against DSB repair pathways. This was done following prior research by Dr. Suzanne Lee at WWU indicating that when these genes were knocked out, there was an accumulation of DSBs
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If the expected results, from running the DNA on the gel were to have happened, what would you continue to do after the future directions
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We would likely verify that Twi7 indeed was upregulated following the induction of Double Stranded Breaks. However, since this result does fall in line with earlier observations by Dr. Suzanne Lee at WWU that Twi7 seems to play less of a role in DSB repair, we likely will not pursue this verification.
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Based on the information gathered within the databases, how were three primers materialized? Did the software create them or did it just guide you to the best primer available for use?
LikeLike
Since the whole Twi7 gene is available on the Tetrahymena genome database, we were able to copy and paste that into the Primer3Plus software. It can autogenerate primer suggestions based on parameters you put into the software. The most important aspects are annealing temperature, GC%, RNA instability, and the risks that it could form secondary structures. We chose the ones that best fit these parameters. The RPPO and Rad51 primers, as well as the validated primers for Twi7 are commercially available.
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