The -20 degree fridge at the lab was left open during the Fourth of July weekend. This was pretty bad because the Phusion enzyme along with the PGem Vector got degraded. I still continued with my PCR but the gel turned out pretty bad. The bandwidths showing up were much smaller than the ones I needed. By Wednesday I still continued the process and after performing transformation and colony PCR, the rat samples were not what I needed them to be. On Thursday I attempted to repeat the nested PCR but that also failed. Thus, I will have to start over on Monday.
At first I was upset with the fridge or whatever made my experiment fail but I now realize that this is science and it may not be my fault. The grad student in my lab, Diana, may have to start over as well, that is two weeks of work wasted. Now that I have completed the full process one time, I will be more comfortable in doing the process again quicker. I will use phusion enzyme for the first reaction. Phusion is harder to deal with but necessary for PGem cloning. I will then use the Taq enzyme for the nested reaction.
Due to the fact that I have repeated these reactions multiple times, I have figured out the optimal temperatures and enzymes. I also understand what I am doing much better because I have to do calculations for PCR by myself and make my own protocols. Below is a map of what I do for sequencing.
I also get to sign packages for the lab, which makes me feel like an equal. The two grad students in my lab always take me to explore places around SF. Usually we drive to a restaurant they like and eat interesting food.
Hopefully the reactions work next week and I can move to the next step of data analysis and DNA Libraries.
.jpeg) |
| Uh Oh. Bad Gel. The bands only go up to the second notch on the ladder (the left most sample). I need about 300 to 400 base pairs for a successful run. This is between the third and fourth band on the ladder. |
At first I was upset with the fridge or whatever made my experiment fail but I now realize that this is science and it may not be my fault. The grad student in my lab, Diana, may have to start over as well, that is two weeks of work wasted. Now that I have completed the full process one time, I will be more comfortable in doing the process again quicker. I will use phusion enzyme for the first reaction. Phusion is harder to deal with but necessary for PGem cloning. I will then use the Taq enzyme for the nested reaction.
Due to the fact that I have repeated these reactions multiple times, I have figured out the optimal temperatures and enzymes. I also understand what I am doing much better because I have to do calculations for PCR by myself and make my own protocols. Below is a map of what I do for sequencing.
.jpeg) |
| It is not very legible but here is an overview of the steps I take to isolate, tag, and sequence the DNA I need from the Rat lymph node samples. |
Hopefully the reactions work next week and I can move to the next step of data analysis and DNA Libraries.
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