6 thoughts on “P35 – Bass

  1. How did you perform the PCR experiment that allowed you to predict that it was a part of the A4 cell cluster?

    1. In order to perform the PCR experiment, we prepared four samples based on our prediction that our phage was part of either the A3 or A4 subcluster, which we determined from the restriction digest. The first sample was a positive control which was made up of DNA that was known to be part of the A3 subcluster and A3 subcluster primers. The second sample was a negative control which was just A3 subcluster primers. The third sample contained diluted DNA we isolated from our phage and A3 subcluster primers. The fourth sample contained diluted DNA we isolated from our phage and A4 subluster primers. All four samples contained PCR reaction mix as well which consists of PCR buffer, all four deoxyribonucleotides, and Taq DNA polymerase. After preparing these samples we ran them in a thermocycler which amplified the DNA if the DNA had regions that matched with the primers. After running the samples in the thermocycler we ran them on a gel. Based on the results of the gel we know that our phage belongs to the A4 subcluster because there was a band present in the lane that had the sample containing our DNA and the A4 subcluster primers, indicating that the primers had matched and the DNA had been amplified. No band present in the lane that had the sample containing our DNA and the A3 subcluster primers, indicates that the primers did not match and there was no DNA amplified.

  2. What is the difference in morphology (head and tail) between phages in the Siphoviridae family and other families of phages?

    1. The Siphoviridae family of phage is characterized by long, noncontractile tails and icosahedral heads. Siphoviridae phage can also have oblong heads with long, noncontractile tails, which can be referred to as a ‘corndog’ phage. The Myoviridae family of phage is characterized by shorter, contractile tails and icosahedral heads. The Podoviridae family of phage is characterized by short, stubby noncontractile tails and icosahedral heads. In all three of these phage families the DNA is stored in the head of the phage, which is also called the capsid, and is made up of proteins. The difference in tail length and contractibility play a role in the way that the phage DNA is injected into the host cell.

  3. What about the results of figure 4 lead you to believe that the phage was part of A3 or A4? What did you expect it to look like, and how did the results compare?

    1. Figure 4 shows a restriction digest which tests samples each containing CutSmart buffer (which increases the effectiveness of restriction enzymes), sterile water, our isolated phage DNA, and one of six restriction enzymes. The restriction enzymes cut DNA at very specific sequences. When the isolated phage DNA is combined with the restriction enzymes and allowed to incubate, the restriction enzymes will look for specific sequences on the DNA that match it’s specific sequence. When a sequence on the DNA matches the sequence from a restriction enzyme, the restriction enzyme will cut the DNA at the location of the match. These cuts from the restriction enzymes appear on the gel as multiple bands or one stretched band seen as unclear on the gel. When running this experiment we didn’t know which restriction enzyme(s), if any, would cut our isolated phage DNA, but we knew that if a band or unclear section appeared then our DNA had been successfully cut. Based on the results from our gel, the restriction enzymes ClaI, HaeIII, and SaII all successfully cut our DNA. From there we compared which enzymes were successful in cutting our DNA with which enzymes were successful in cutting DNA in various other clusters. From this comparison we made the prediction that our phage was in either the A3 or A4 subcluster because the restriction enzymes that successfully cut our DNA also successfully cut DNA in those subclusters and had bands at approximately the same location on the gel as ours did.

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