For the most part, our lab ran smoothly. At one point we experienced contamination in our phage buffer, but we only had to redo that specific experiment once. Another error we ran into was during our PCR (shown in figure 4), as our control showed no results so we knew there was an error in either mixing with the primers or running on the gel. Overall, though, we were very successful in our lab and were able to complete the entire protocol.
DNA sequencing can confirm some of the results explained on the poster. For example, the restriction digest gave us an idea of our phage’s cluster, which led us to “confirm” our phage to be in the sub cluster F1 from the PCR experiment. In reality, while these experiments are useful, there is no way to 100% confirm results like the cluster categorization without sequencing.
The plate in figure 2 was incubated at 37 degrees for approximately 48 hours. From my knowledge and the results of our experiments, once a plate has incubated for at least 24 hours there is not likely to be much change in the results post that period. Plates from the beginning of the semester were kept until just recently and showed no differences.
Yes, we used one positive control for our PCR. The second lane from the left in figure 4 served as the control. This was composed of isolated phage DNA from a different, sequenced phage. That DNA was mixed with a primer from the sub-cluster it was known to be a part of. This means that the primer should successfully amplify the DNA and we should be able to see a bright band formed on the gel. If no band is seen, this means there was some experimental or human error that caused the PCR experiment to be flawed.
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Did your lab run smoothly or did you run into any human errors that lead you to having to redo the gel?
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For the most part, our lab ran smoothly. At one point we experienced contamination in our phage buffer, but we only had to redo that specific experiment once. Another error we ran into was during our PCR (shown in figure 4), as our control showed no results so we knew there was an error in either mixing with the primers or running on the gel. Overall, though, we were very successful in our lab and were able to complete the entire protocol.
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What is learned from a DNA sequence, and how would the results of a DNA sequencing affect your future direction?
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DNA sequencing can confirm some of the results explained on the poster. For example, the restriction digest gave us an idea of our phage’s cluster, which led us to “confirm” our phage to be in the sub cluster F1 from the PCR experiment. In reality, while these experiments are useful, there is no way to 100% confirm results like the cluster categorization without sequencing.
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How long was the plate in Figure 2 incubated for to acquire these results? Would the results have changed if it was longer/shorter?
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The plate in figure 2 was incubated at 37 degrees for approximately 48 hours. From my knowledge and the results of our experiments, once a plate has incubated for at least 24 hours there is not likely to be much change in the results post that period. Plates from the beginning of the semester were kept until just recently and showed no differences.
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Did you have controls when you ran your PCR? If so, what were they, and why were they used?
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Yes, we used one positive control for our PCR. The second lane from the left in figure 4 served as the control. This was composed of isolated phage DNA from a different, sequenced phage. That DNA was mixed with a primer from the sub-cluster it was known to be a part of. This means that the primer should successfully amplify the DNA and we should be able to see a bright band formed on the gel. If no band is seen, this means there was some experimental or human error that caused the PCR experiment to be flawed.
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