7 thoughts on “P13 – Canales

  1. What is a restriction digest? Could you please explain the detailed protocol for how this was done?

    1. Restriction digest is a process to separate DNA into fragments. I used 6 different restriction enzymes to prepare 6 different samples that consisted of 10 U of a particular restriction enzyme, 2 µL of my phage DNA, 3 µL 1X CutSmart buffer, 25 µL sterile water, and 1X loading dye. In total I had 7 samples, the 6 samples I described above and a control that consisted of 2 µL of our phage DNA, 3 µL 1X CutSmart buffer, 25 µL sterile water and 1X loading dye. After creating the samples we incubated them for 48 hours and then proceed to load them into a 0.8% agarose gel to then run a gel electrophoresis. After the gel was run I took it to the UV light source to see the results on the gel. This was done to help visualize our DNA fragments to then later find out which cluster they are in based on which enzymes cut our DNA.

  2. Do you have any ideas as to how your phage lysate was contaminated? How did you determine your contaminating phage was Corndog?

    1. I am still not completely sure how my phage lysate was contaminated. It could’ve been our top agar or our LB media that I used in a lot of our primary experiments or some other shared substance we may have used in lab, since multiple people in my lab section had the contaminate as well. The main thing that helped me determine that I did have a contaminate phage was my electron microscopy image that showed both Moonpie and Corndog present in the images. After consulting with my TA, she informed me that I did in fact have the contaminate and also my PCR showed cluster O, which I later found out is Corndog’s cluster.

    1. First, be very careful when working in your sterile area, as well as double checking with your TAs and LAs that no contamination is present in anything you may be preparing for you ahead of time. You must have the flame turned on when working on most of these experiments and it will create a pretty sterile area. Also make sure your pipettes and other tools don’t touch your clothes or the bench because contamination can happen very easily if we are not careful. You just basically have to be careful and diligent when going through all of the experiments in this process. You can confirm that a contaminate phage is present mainly through the electron microscopy image since it is able to give you images we can not see with our naked eyes.

    2. The main thing that will confirm if a contaminate phage is present along with the phage your are trying to isolate is through the electron microscopy image. If more than one kind of phage species is present, in the EM picture they will both show up. Also if you have contamination in your top agar or LB media, your plaque assays will visibly be contaminated since it won’t be a uniform lawn of agar all across the L-plate.

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