Hello everyone! I just got back from Chicago in time to update my blog for this week. I wasn't able to do too much being out of state but I was able to obtain some results. First off, that pesky BL-21 that refused to grow the first few times final decided to grow.
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| Two different batches of BL-21 were used, both worked. |
The problem may have come from the BL-21 that I was using, as I used a new batch of cells for this transformation. Next week, I will be working to grow more of the BL-21 and purify the pfu that it is producing.
I was also able to extract the plasmid that I had used originally on the DH5α cells and run a gel electrophoresis on it.
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| Two separate batches of plasmid were used here with a marker in the middle. |
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To perform the gel, I had to mix the plasmid with restriction enzymes, a protein which cleaves DNA at specific sites. This is called a restriction digest. From the picture above, each row shows a different digestions or non-digestions with a marker in the middle to show us the length of DNA segments. The bands that are furthest behind are the undigested controls, the two below that are only digested by a single enzyme, while the last ones are digested by both enzymes, and thus have 2 bands of varying lengths. With this, I can determine what plasmid I used because we were not entirely sure to begin with.
Stay tuned in for next week, where I will attempt to purify the pfu from the BL-21!
I'm glad you're finally able to do the gel electrophoresis that we only read about in AP Biology. I'm sure seeing is a lot more insightful than just reading about it. Why does using a new batch of cells pose a problem for culturing? Is it just that the cells need to mature for a certain amount of time?
ReplyDeleteIt's kind of fun seeing what we learned in bio coming to help me understand more of what I am doing. Really the only problem with using new cells is the growth time. A lot of the time in the lab, I am working on something else in order to wait for something I was working on previously to be ready.
Deletehi dylan,
ReplyDeleteCan you say something about your new workplace? How do you like being in the lab and out of the classroom?
Mr. Bloom
The workplace is really nice. All the other researchers are kind, though I do not speak to them much as the lab is split into specific areas, where only a few people work in the same area as me, like a grad student who has been helping me with my experiments. As for being in a lab as opposed to a classroom, it feels a lot more "free", though there is a lot more room for error.
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ReplyDeleteHey Dylan! Sounds like so much fun over there! I'm so jealous that you get to participate in all these processes we discusses in AP Bio! Just to refresh my memory in the gel electrophoresis picture the top is to the right, correct?
ReplyDeleteYes, the DNA has started on the right in those little rectangular columns (which was a pain to get the DNA is there because it's about 0.5 mm thick) and has now flowed through the gel to the left side.
DeleteDylan, your project looks so exciting! I'm glad the BL-21 finally grew. Are you going to investigate more about why the original BL-21 didn't grow?
ReplyDeleteIt's kinda hard to figure out why the original would not grow because we are out of stock of the original BL21s (go figure), but its not terribly important to the experiment as a whole, unless these BL21s that i grew do not grow again if I were to plate them once more.
DeleteHi Dylan! I'm very happy that your experiments are working out well! It is so weird to see the picture of the gel electrophoresis outside of an AP textbook! What was the procedure like for this? Did you perform this procedure?
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